Method of treating inflammation with statins

ABSTRACT

The current invention discloses novel methods for the inhibition of inducible nitric oxide synthesis (iNOS) and the production of NO. Methods of inhibiting the induction of proinflammatory cytokines are also described. Methods of treating various disease states, such as X-linked adrenoleukodystrophy, multiple sclerosis, Alzheimer&#39;s and septic shock using inhibitors of iNOS and cytokine induction are disclosed. The inhibitors include the exemplary compounds lovastatin, a sodium salt of phenylacetic acid (NaPA), FPT inhibitor II, N-acetyl cysteine (NAC), and cAMP.

This application is a continuation of application Ser. No. 12/145,261,which was filed on Jun. 24, 2008 now abandoned, as a continuationapplication of U.S. application Ser. No. 11/204,288, which was filed onAug. 15, 2005 (now issued as U.S. Pat. No. 7,396,659), which is acontinuation application of U.S. application Ser. No. 10/273,557, filedon Oct. 18, 2002 (now issued as U.S. Pat. No. 7,049,058), which is adivisional of U.S. application Ser. No. 09/579,791 filed May 25 2000(now issued as U.S. Pat. No. 6,511,800) which is a continuation ofInternational Application No. PCT/US98/25360 filed Nov. 25, 1998, whichclaims the benefit of U.S. Provisional Application No. 60/066,839 filedNov. 25, 1997. The entire text of the aforementioned applications isincorporated herein by reference in its entirety.

This invention was made with government support under Grant No. NS-22576awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the treatment of conditionsinvolving undesired or pathological levels of inducible nitric oxidesynthase (iNOS), e.g. septic shock or neuroinflammatory diseases. In oneimportant aspect, the invention relates to methods of suppressing,inhibiting or preventing the accumulation of nitric-oxide inducedcytotoxicity by using inhibitors that block or suppress the induction ofcytokines and/or inducible nitric oxide synthase. Another aspect of theinvention is the treatment of conditions involving undesired orpathological levels of proinflammatory cytokines (i.e. TNF-α, IL-1β,IL-2, IL-6, IL-8 and/or IFN-γ) and/or iNOS. One important aspect of theinvention relates to methods of suppressing, inhibiting, or preventingproinflammatory cytokines and/or iNOS induced or aggravated disordersincluding conditions involving the detrimental effects of inflammation(e.g. disorders such as lupus, rheumatoid arthritis, osteoarthritis,amyotrophic lateral sclerosis, and autoimmune disorders;ischemia/reperfusion; neuroinflammatory conditions such as Alzheimer's,stroke, multiple sclerosis, X-linked adrenoleukodystrophy; and theeffects of aging).

2. Description of Related Art

Nitric Oxide and Proinflammatory Cytokines

Nitric oxide (NO) is a potent pleiotropic mediator of physiologicalprocesses such as smooth muscle relaxation, neuronal signaling,inhibition of platelet aggregation and regulation of cell mediatedtoxicity. It is a diffusible free radical which plays many roles as aneffector molecule in diverse biological systems including neuronalmessenger, vasodilation and antimicrobial and antitumor activities(Nathan, 1992; Jaffrey et al., 1995). NO appears to have both neurotoxicand neuroprotective effects and may have a role in the pathogenesis ofstroke and other neurodegenerative diseases and in demyelinatingconditions (e.g. multiple sclerosis, experimental allergicencephalopathy, X-adrenoleukodystrophy) and in ischemia and traumaticinjuries associated with infiltrating macrophages and the production ofproinflamatory cytokines (Mitrovic et al., 1994; Bo et al., 1994;Merrill et al., 1993; Dawson et al., 1991, Kopranski et al., 1993;Bonfoco et al., 1995). A number of pro-inflammatory cytokines andendotoxin (bacterial lipopolysaccharide, LPS) also induce the expressionof iNOS in a number of cells, including macrophages, vascular smoothmuscle cells, epithelial cells, fibroblasts, glial cells, cardiacmyocytes as well as vascular and non-vascular smooth muscle cells.Although monocytes/macrophages are the primary source of iNOS ininflammation, LPS and other cytokines induce a similar response inastrocytes and microglia (Hu et al., 1995; Galea et al., 1992).

During inflammation, reactive oxygen species (ROS) are generated byvarious cells including activated phagocytic leukocytes; for example,during the neutrophil “respiratory burst”, superoxide anion is generatedby the membrane-bound NADPH oxidase. ROS are also believed to accumulatewhen tissues are subjected to inflammatory conditions including ischemiafollowed by reperfusion. Superoxide is also produced under physiologicalconditions and is kept in check by superoxide dismutates. Excessivelyproduced superoxide overwhelms the antioxidant capacity of the cell andreacts with NO to form peroxynitrite, ONOO⁻, which may decay and giverise to hydroxyl radicals, OH (Marietta, M., 1989; Moncada et al., 1989;Saran et al., 1990; Beckman et al. 1990). NO, peroxynitrite and OH arepotentially toxic molecules to cells including neurons andoligodendrocytes that may mediate toxicity through modification ofbiomolecules including the formation of iron-NO complexes of ironcontaining enzyme systems (Drapier et al., 1988), oxidation of proteinsulfhydryl groups (Radi et al., 1991), nitration of proteins andnitrosylation of nucleic acids and DNA strand breaks (Wink et al.,1991).

There is now substantial evidence that iNOS plays an important role inthe pathogenesis of a variety of diseases. In addition, it is nowthought that excess NO production may be involved in a number ofconditions, including conditions that involve systemic hypotension suchas septic and toxic shock and therapy with certain cytokines.Circulatory shock of various etiologies is associated with profoundchanges in the body's NO homeostasis. In animal models of endotoxicshock, endotoxin produces an acute release of NO from the constitutiveisoform of nitric oxide synthase in the early phase, which is followedby induction of iNOS. NO derived from macrophages, microglia andastrocytes has been implicated in the damage of myelin producingoligodendrocytes in demyelinating disorders like multiple sclerosis andneuronal death during neuronal degenerating conditions including braintrauma (Hu et al., 1995; Galea et al., 1992; Koprowski et al., 1993;Mitrovic et al., 1994; Bo et al., 1994; Merrill et al., 1993).

NO is synthesized from L-arginine by the enzyme nitric oxide synthase(NOS) (Nathan, 1992). Nitric oxide synthases are classified into twogroups. One type, constitutively expressed (cNOS) in several cell types(e.g., neurons, endothelial cells), is regulated predominantly at thepost-transcriptional level by calmodulin in a calcium dependent manner(Nathan, 1992; Jaffrey et al., 1995). In contrast, the inducible form(iNOS), synthesized de novo in response to different stimuli in variouscell types including macrophages, hepatocytes, myocytes, neutrophils,endothelial and messangial cells, is independent of calcium. Astrocytes,the predominant glial component of brain have also been shown to induceiNOS in response to bacterial lipopolysaccharide (LPS) and a series ofproinflammatory cytokines including interleukin-1β (IL-1β), tumornecrosis factor-α (TNF-α), interferon-γ (IFN-γ) (Hu et al., 1995; Galeaet al., 1992).

Cytokines associated with extracellular signaling are involved in thenormal process of host defense against infections and injury, inmechanisms of autoimmunity and in the pathogenesis of chronicinflammatory diseases. It is believed that nitric oxide (NO),synthesized by nitric oxide synthetase (NOS) mediates deleteriouseffects of the cytokines (Nathan, 1987; Zang et al., 1993; Kubes et al.,1991). For example, NO as a result of stimuli by cytokines (e.g., TNF-α,IL-1 and interleukin-6 (IL-6) is implicated in autoimmune diseases suchas multiple sclerosis, rheumatoid arthritis, osteoarthritis (Zang etal., 1993; McCartney-Francis et al., 1993). The NO produced by iNOS isassociated with bactericidal properties of macrophages (Nathan, 1992;Stuehr et al., 1989). Recently, an increasing number of cells (includingmuscle cells, macrophages, keratinocytes, hepatocytes and brain cells)have been shown to induce iNOS in response to a series ofproinflammatory cytokines including IL-1, TNF-α, interferon-γ (IFN-γ)and bacterial lipopolysaccharides (LPS) (Zang et al., 1993; Busse etal., 1990; Genge et al., 1995).

Signal Transduction Pathways

Mevalonate metabolites, particularly farnesyl pyrophosphate (FPP), areinvolved in post-translational modification of some G-proteins,including Ras (Goldstein et al., 1990; Casey et al., 1989). Theinhibition of isoprenylation of Ras proteins by inhibitors of mevalonatepathway and their membrane association and transduction of signal fromRas to Raf/MAP kinase cascade (Kikuchi et al., 1994) indicates a role ofmevalonate metabolites in the transduction of signal from receptortyrosine kinases to Raf/MAP kinase cascade. Two enzymes that control therate-limiting steps of the mevalonate pathway are3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, whichcatalyzes the formation of mevalonate from acetyl-CoA, and mevalonatepyrophosphate decarboxylase, which controls the use of mevalonate withinthe cell by converting 3-phospho-5-pyrophospho-mevalonate to isopentenylpyrophosphate. Lovastatin, a potent inhibitor of HMG-CoA reductase, andsodium salt of phenylacetic acid (NaPA), an inhibitor of mevalonatepyrophosphate decarboxylase, are known to reduce the level of cellularisoprenoids (Castillo et al., 1991; Samid et al., 1994) andisoprenylated proteins (Repko and Maltese, 1989). No suppression ofisoprenylated protein maturation in vitro by lovastatin treatment thatproduced 50% inhibition of sterol biosynthesis has been observed(Sinensky et al., 1991). The IC₅₀ for inhibition of sterol synthesis is10 nM, whereas the IC₅₀ for inhibition of conversion of pro-p21^(ras) tomature-p21^(ras) is maximal at 2.6 μM (Sinensky et al., 1991). Thepharmacologically attainable concentration for NaPA, however, is 1 to 5mM (Thibault et al., 1995). HMG-CoA reductase can also be inhibited by5-amino 4-imidazolecarboxamide ribotide (AICAR). AICAR stimulatesAMP-activated protein kinase, an enzyme that inhibits acetyl-CoAcarboxylase and HMG-CoA reductase (Henin et al., 1995)

LPS is shown to bind cell-surface receptor CD14 (Stefanova et al., 1993)and induce iNOS, probably via activation of NFkβ (Xie et al., 1994; Kwonet al., 1995). NFkβ is an ubiquitous multisubunit transcription factorthat is activated in response to various stimuli including cytokinesTNF-α, IL-1, IL-2, IL-6, viruses, LPS, DNA damaging agents and phorbolmyristate acetate (PMA) (Schreck et al., 1992). Previous studies (Law etal., 1992) demonstrating the inhibition of NF-kβ activation by mevinolinand 5′-methylthioadenosine indicates a role of protein farnesylation andcarboxyl methylation reactions in the activation of NF-kβ.Identification of the binding site of NF-kβ in the promoter region ofthe iNOS gene and that the activation of NFkβ in LPS-induced iNOSinduction establishes a role of NFkβ activation in the induction of iNOS(Xie et al., 1994). Although the precise mechanism of NFkβ activation isnot known at the present time, the inhibition of activation of NFkβ byinhibitors of tyrosine kinase and proteases indicates a role ofphosphorylation and degradation of Ikβ in this process (Menon et al.,1993; Henkel et al., 1993).

Reactive oxygen (Schreck et al., 1992) and reactive nitrogen (Lander etal., 1993) species have been demonstrated to mediate the signal for NFkβactivation. The differential induction of NFkβ by protein phosphataseinhibitors in primary and transformed cell lines also indicates thatinduction of NFkβ is dependent on the dual processes of cellular redoxand phosphorylation (Menon et al., 1993). The exact target of ROS thatmodulate cellular redox is unknown, and the lack of induction in cellsin which activity of p21ras was blocked through expression of a dominantnegative mutant or treatment with farnesyltransferase inhibitor indicatethat direct activation of p21ras may be the central mechanism by whichredox stress stimuli transmit its signal to the nucleus (Lander et al.,1995).

Ceramide Production and Apoptosis

Cytokine-mediated ceramide production is implicated in apoptosis ofdifferent cells including brain cells (Brugg et al., 1996; Wiesner andDawson, 1996). Several studies support a role for hydrolysis ofsphingomyelin as a stress-activated signaling mechanism in whichceramide plays a role in cell regulation, cell differentiation, growthsuppression and apoptosis in various cell types including glial andneuronal cells (Hannun and Bell, 1989; Hannun, 1994; Kolesnick andGolde, 1994; Brugg et al., 1996; Wiesner and Dawson, 1996).Sphingomyelin is preferentially concentrated in the outer leaflet of theplasma membrane of most mammalian cells; it comprises sphingosine (along chain sphingoid base backbone), a fatty acid, and a phosphocholinehead group. Ceramide is composed of a sphingoid base with a fatty acidin amide linkage. Ceramide activates the proteases of theinterleukin-converting enzyme (ICE) family (especiallyprICE/YAMA/CPP32), the protease responsible for cleavage ofpoly(A)DP-ribose polymerase (Martin et al., 1995), and that theactivation of prICE by ceramide and induction of apoptosis are inhibitedby overexpression of Bcl-2 (Zhang et al., 1996). Addition of exogenousceramides or sphingomyelinase to cells induces stress-activated proteinkinase-dependent transcriptional activity through the activation ofc-jun (Latinis and Koretzky, 1996), and a dominant negative mutant ofSEK1, the protein kinase responsible for phosphorylation and activationof stress-activated protein kinase, interferes with ceramide-inducedapoptosis (Verheij et al., 1996). These observations also indicate thatboth Bcl-2 and stress-activated protein kinase function downstream ofceramide in the apoptotic pathway.

The signaling events in cytokine-mediated activation of sphingomyelindegradation to ceramide are poorly understood. Since the discovery ofthe sphingomyelin cycle, several inducers have been shown to be coupledto sphingomyelin-ceramide signaling events, including1α,25-dihydroxyvitamin D₃, radiation, antibody cross-linking, TNF-α,IFN-γ, IL-1β, nerve growth factor, and brefeldin A (Hannun and Bell,1989; Hannun, 1994; Kolesnick and Golde, 1994; Zhang and Kolesnick,1995; Kantey et al., 1995; Linardic et al., 1996).

The sphingomyelin pathway-associated signal transduction pathwaymediates the action of several extracellular stimuli that lead toimportant biochemical and cellular effects (Zhang and Kolesnick, 1995;Kantey et al., 1995; Yao et al., 1995; Hannun, 1996; Lozano et al.,1994). In the case of TNF-α, the pathway is initiated by the action ofTNF-α on its 55-kDa receptor, leading to phospholipase A₂ activation,generation of arachidonic acid, and subsequent activation ofsphingomyelinase (Jayadev et al., 1994). This pathway is initiated bythe activation of two distinct forms of sphingomyelinase (SMase), amembrane-associated neutral sphingomyelinase (Chatterjee, 1993) and anacidic sphingomyelinase (Spence, 1993), which reside in the caveola andthe endosomal-lysosomal compartment. Each type of SMase hydrolyzes thephosphodiester bond of sphingomyelin to yield ceramide andphosphocholine. Proinflammatory cytokines (tumor necrosis factor-α,TNF-α; interleukin-1β, IL-1β; interferon-γ, IFN-γ) and bacteriallipopolysaccharides have been shown as potent inducers of SMases.Ceramide has emerged as a second messenger molecule that is consideredto mimic most of the cellular effects of cytokines andlipopolysaccharide in terminal differentiation, apoptosis, and cellcycle arrest (Zhang and Kolesnick, 1995; Kantey et al., 1995).

Sphingomyelin turnover and ceramide generation in response to TNF-α andIL-1β, occurs within minutes of stimulation; however, the sequence ofevents linking receptor stimulation and SMase activation remains largelyunknown (Hannun, 1996; Lozano et al., 1994; Jayadev et al., 1994). In anumber of cell systems, interaction of TNF-α with its membrane receptors(p75 and p55) has been found to activate phospholipase A₂ and to inducerelease of arachidonic acid from phosphatidylcholine andphosphatidylethanolamine pools. This arachidonic acid has been shown asa mediator of sphingomyelin hydrolysis in response to TNF-α (Jayadev etal., 1994). In addition, proteases have also been implicated in thepathway leading from TNF-α to the activation of SMase (Hannun, 1996;Dbaio et al., 1997) recently. On the other hand, IL-1β and TNF-α areknown to induce the production of reactive oxygen species, a class ofhighly diffusible and ubiquitous molecules, which have been suggested toact as second messengers (Tiku et al., 1990; Lo and Cruz, 1995; Devaryet al., 1991). ROS encompassing species such as superoxide, hydrogenperoxide, and hydroxyl radicals are known to regulate critical steps inthe signal transduction cascade and many important cellular eventsincluding protein phosphorylation, gene expression, transcription factoractivation, DNA synthesis, and cellular proliferation (Schreck et al.,1991; Sen and Packer, 1996). A recent observation has shown thatglutathione or similar molecules inhibit the activity ofmagnesium-dependent neutral SMase in vitro (Liu and Hannun, 1997).However, surprisingly, the SH group of GSH was not required as S-methylGSH and GSSG inhibited neutral SMase at lower concentrations than GSH(Liu and Hannun, 1997). On the other hand, N-acetylcysteine has alsobeen found to inhibit the synthesis of ceramide in cultured rathepatocytes through the inhibition of dihydroceramide desaturase (Michelet al., 1997).

Inflammatory Diseases

NO generated by iNOS has been implicated in the pathogenesis ofinflammatory diseases. In experimental animals hypotension induced byLPS or TNF-alpha can be reversed by NOS inhibitors and reinitiated byL-arginine (Kilbourn et al., 1990). Conditions which lead tocytokine-induced hypotension include septic shock, hemodialysis (Beasleyand Brenner, 1992) and IL-2 therapy in cancer patients (Hibbs et al.,1992). Studies in animal models have suggested a role for NO in thepathogenesis of inflammation and pain and NOS inhibitors have been shownto have beneficial effects on some aspects of the inflammation andtissue changes seen in models of inflammatory bowel disease (Miller etal., 1990) and cerebral ischemia and arthritis (Ialenti et al., 1993;Stevanovic-Racic et al., 1994).

Inflammation, iNOS activity and/or cytokine production has beenimplicated in a variety of diseases and conditions, including psoriasis(Ruzicka et al., 1994; Kolb-Bachofen et al., 1994; Bull et al., 1994);uveitis (Mandia et al., 1994); type 1 diabetes (Eisieik & Leijersfam,1994; Kroncke et al., 1991; Welsh el al., 1991); septic shock (Petros etal., 1991; Thiemermann & Vane, 1992; Evans et al., 1992; Schilling etal., 1993); pain (Moore et al., 1991; Moore et al, 1992; Meller et al.,1992; Lee et al., 1992); migraine (Olesen et al., 1994); rheumatoidarthritis (Kaurs & Halliwell, 1994); osteoarthritis (Stadler et al.,1991); inflammatory bowel disease (Miller et al., 1993; Miller et al.,1993); asthma (Hamid et al., 1993; Kharitonov et al., 1994); Koprowskiet al., 1993); immune complex diseases (Mulligan et al., 1992); multiplesclerosis (Koprowski et al., 1993); ischemic brain edema (Nagafuji etal., 1992; Buisson et al., 1992; Trifiletti et al., 1992); toxic shocksyndrome (Zembowicz & Vane, 1992); heart failure (Winlaw et al., 1994);ulcerative colitis (Boughton-Smith et al., 1993); atherosclerosis (Whiteet al., 1994); glomerulonephritis (Muhl et al., 1994); Paget's diseaseand osteoporosis (Lowick et al., 1994); inflammatory sequelae of viralinfections (Koprowski et al., 1993); retinitis, (Goureau et al., 1992);oxidant induced lung injury (Berisha et al., 1994); eczema (Ruzica etal., 1994); acute allograft rejection (Devlin, J. et al., 1994); andinfection caused by invasive microorganisms which produce NO (Chen, Yand Rosazza, J. P. N., 1994).

In the central nervous system, apoptosis may play an importantpathogenetic role in neurodegenerative diseases such as ischemic injuryand white matter diseases (Thompson, 1995; Bredesen, 1995). BothX-linked adrenoleukodystrophy (X-ALD) and multiple sclerosis (MS) aredemyelinating diseases with the involvement of proinflammatory cytokinesin the manifestation of white matter inflammation. The presence ofimmunoreactive tumor necrosis factor a (TNF-α) and interleukin 1 (IL-1β)in astrocytes and microglia of X-ALD brain has indicated the involvementof these cytokines in immunopathology of X-ALD and aligned X-ALD withMS, the most common immune-mediated demyelinating disease of the CNS inman (Powers, 1995; Powers et al., 1992; McGuiness et al., 1995;McGuiness et al., 1997). Several studies demonstrating the induction ofproinflammatory cytokines at the protein or mRNA level in cerebrospinalfluid and brain tissue of MS patients have established an association ofproinflammatory cytokines (TNF-α, IL-1β, IL-2, IL-6, and IFN-γ) with theinflammatory loci in MS (Maimone et al., 1991; Tsukada et al., 1991;Rudick and Ransohoff, 1992).

X-linked adrenoleukodystrophy (X-ALD), an inherited, recessiveperoxisomal disorder, is characterized by progressive demyelination andadrenal insufficiency (Singh, 1997; Moser et al., 1984). It is the mostcommon peroxisomal disorder affecting between 1/15,000 to 1/20,000 boysand manifests with different degrees of neurological disability. Theonset of childhood X-ALD, the major form of X-ALD, is between the age of4 to 8 and then death within the next 2 to 3 years. Although X-ALDpresents as various clinical phenotypes, including childhood X-ALD,adrenomyeloneuropathy (AMN), and Addison's disease, all forms of X-ALDare associated with the pathognomonic accumulation of saturated verylong chain fatty acids (VLCFA) (those with more than 22 carbon atoms) asa constituent of cholesterol esters, phospholipids and gangliosides(Moser et al., 1984) and secondary neuroinflammatory damage (Moser etal., 1995). The necrologic damage in X-linked adrenoleukodystrophy maybe mediated by the activation of astrocytes and the induction ofproinflammatory cytokines. Due to the presence of similar concentrationof VLCFA in plasma and as well as in fibroblasts of X-ALD, fibroblastsare generally used for both prenatal and postnatal diagnosis of thedisease (Singh, 1997; Moser et al., 1984).

The deficient activity for oxidation of lignoceroyl-CoA ligase ascompared to the normal oxidation of lignoceroyl-CoA in purifiedperoxisomes isolated from fibroblasts of X-ALD indicated that theabnormality in the oxidation of VLCFA may be due to deficient activityof lignoceroyl-CoA ligase required for the activation of lignoceric acidto lignoceroyl-CoA (Hashmi et al., 1986; Lazo et al., 1988). While thesemetabolic studies indicated lignoceroyl-CoA ligase gene as a X-ALD gene,positional cloning studies led to the identification of a gene thatencodes a protein (ALDP), with significant homology with the ATP-bindingcassette (ABC) of the super-family of transporters (Mosser et al.,1993). The normalization of fatty acids in X-ALD cells followingtransfection of the X-ALD gene (Cartier et al., 1995) supports a rolefor ALDP in fatty acid metabolism; however, the precise function of ALDPin the metabolism of VLCFA is not known at present.

Similar to other genetic diseases affecting the central nervous system,the gene therapy in X-ALD does not seem to be a real option in the nearfuture and in the absence of such a treatment a number of therapeuticapplications have been investigated (Singh, 1997; Moser, 1995). Adrenalinsufficiency associated with X-ALD responds readily with steroidreplacement therapy, however, there is as yet no proven therapy forneurological disability (Moser, 1995). Addition of monoenoic fatty acid(e.g., oleic acid) to cultured skin fibroblasts of X-ALD patients causesa reduction of saturated VLCFA presumably by competition for the samechain elongation enzyme (Moser, 1995). Treatment of X-ALD patients withtrioleate resulted in 50% reduction of VLCFA. Subsequent treatment ofX-ALD patients with a mixture of trioleate and trieruciate (popularlyknown as Lorenzo's oil) also led to a decrease in plasma levels of VLCFA(Moser, 1995; Rizzo et al., 1986; Rizzo et al., 1989). Unfortunately,the clinical efficacy has been unsatisfactory since no proof offavorable effects has been observed by attenuation of the myelinolyticinflammation in X-ALD patients (Moser, 1995). Moreover, the exogenousaddition of unsaturated VLCFA induces the production of superoxide, ahighly reactive oxygen radical, by human neutrophils (Hardy et al.,1994). Since cerebral demyelination of X-ALD is associated with a largeinfiltration of phagocytic cells to the site of the lesion (Powers etal., 1992), treatment with unsaturated fatty acids may even be toxic toX-ALD patients. Bone marrow therapy also appears to be of only limitedvalue because of the complexity of the protocol and of insignificantefficacy in improving the clinical status of the patient (Moser, 1995).

Experimental allergic encephalomyelitis (EAE) is an inflammatorydemyelinating disease of the central nervous system (CNS) that serves asa model for the human demyelinating disease, multiple sclerosis (MS).Studies have shown that the majority of the inflammatory cellsconstitute of T-lymphocytes and macrophages (Merrill and Benveniste,1996). These effector cells and astrocytes have been implicated in thedisease pathogenesis by secreting number of molecules that act asinflammatory mediators and/or tissue damaging agents such as nitricoxide (NO). NO is a molecule with beneficial as well as detrimentaleffects. In neuroinflammatory diseases like EAE, high amounts of NOproduced for longer durations by inducible nitric oxide synthase (iNOS)acts as a cytotoxic agent towards neuronal cells. Previous studies haveshown NO by itself or it's reactive product (ONOO⁻) may be responsiblefor death of oligodendrocytes, the myelin producing cells of the CNS,and resulting in demyelination in the neuroinflammatory diseaseprocesses (Merrill et al., 1993; Mitrovic et al., 1994).

Infiltrating T-lymphocytes in EAE produce pro-inflammatory cytokinessuch as IL-12, TNF-α and IFN-γ (Merrill and Benveniste, 1996). Inaddition to T-cells and macrophages, astrocytes have also been shown toproduce TNF-α (Shafer and Murphy, 1997). Convincing evidence exists tosupport a role for both TNF-α and IFN-γ in the pathogenesis of EAE(Taupin et al., 1997; Villarroya et al., 1996; Issazadeh et al., 1995).Investigations with antibodies against TNF-α have shown that in micethese antibodies protect against active and adaptively transferred EAEdisease (Klinkert et al., 1997). The expression of TNF-α and IFN-γduring EAE disease could result in the upregulation of iNOS inmacrophage and astrocytes because TNF-α and IFN-γ have been shown to bepotent inducers of iNOS in macrophages and astrocytes in culture (Xie etal., 1994). This induction of iNOS could result in the production of NO,which if produced in large amounts may lead to cytotoxic effects.Peroxynitrite (ONOO⁻) has been identified in both MS and EAE CNS (Hooperet al., 1997; van der Veen et al., 1997). The role of peroxynitrite inthe pathogenesis of EAE is supported by the beneficial effects of uricacid, a peroxynitrite scavenger, against EAE and by a subsequent surveydocumenting that MS patients had significantly lower serum uric acidlevels than those of controls (Hooper et al., 1998). However,aggravation of EAE by inhibitors of NOS activity (Ruuls et al., 1996)and in an animal model of iNOS gene knockout (Fenyk-Melody et al., 1998)indicate that NO may not be the only pathological mediator in EAEdisease process. In addition to NO other free radicals such as reactiveoxygen intermediates (O₂ ⁻, H₂O₂, and OH⁻) can also be stimulated bycytokines (Merrill and Benveniste, 1996). Reactive oxygen intermediates(ROI) and NO are believed to be key mediators of pathophysiologicalchanges that take place during inflammatory disease process. ROI's suchas superoxide anion, hydroxy radicals and hydrogen peroxide can also bestimulated by TNF-α (Merrill and Benveniste, 1996). Therefore, it islikely that both the direct modulation of cellular functions byproinflammatory cytokines and toxicity of the ROI and reactive nitrogenspecies may play a role in the pathogenesis of EAE disease.

Several studies on protein and/or mRNA levels in plasma, cerebrospinalfluid (CSF), brain tissue, and cultured blood leukocytes from MSpatients have established an association of proinflammatory cytokines(TNF-α, IL-1 and IFN-γ) with MS (Taupin et al., 1997; Villarroya et al.,1996; Issazadeh et al., 1995). The mRNA for iNOS has also beendetectable in both MS as well as EAE brains (Bagasra et al., 1995;Koprowski et al., 1993). Semiquantitative RT-PCR™ for iNOS mRNA in MSbrains shows markedly higher expression of iNOS mRNA in MS brains thancontrol brains (Bagasra et al., 1995). Analysis of CSF from MS patientshas also shown increased levels of nitrite and nitrate compared withnormal control (Merrill and Benveniste, 1996). Peroxynitrite, ONOO— is astrong nitrosating agent capable of nitrosating tyrosine residues ofproteins to nitrotyrosine. Increased levels of nitrotyrosine have beenfound in demyelinating lesions of MS brains as well as spinal cords ofmice with EAE (Hooper et al., 1998; Hooper et al., 1997). A strongcorrelation exists between CSF levels of cytokines, disruption ofblood-brain barrier, and high levels of circulating cytokines in MSpatients (Villarroya et al., 1996; Issazadeh et al., 1995). Increase inTNF-α and IFN-γ levels seems to predict relapse in MS and the number ofcirculating IFN-γ positive blood cells correlates with severity ofdisability. These observations support the view that in both MS and EAE,induction of proinflammatory cytokines and production of NO through iNOSplay roles in the pathogenesis of these diseases.

Alzheimer's disease (AD) is the most common degenerative dementiaaffecting primarily the elderly population. The disease is characterizedby the decline of multiple cognitive functions and a progressive loss ofneurons in the central nervous system. Deposition of beta-amyloidpeptide has also been associated with AD. Over the last decade, a numberof investigators have noted that AD brains contain many of the classicalmarkers of immune mediated damage. These include elevated numbers ofmicroglia cells, which are believed to be an endogenous CNS form of theperipheral macrophage, and astrocytes. Of particular importance,complement proteins have been immunohistochemically detected in the ADbrain and they most often appear associated with beta-amyloid containingpathological structures known as senile plaques (Rogers et al., 1992;Haga et al., 1993).

These initial observations which suggest the existence of aninflammatory component in the neurodegeneration observed in AD has beenextended to the clinic. A small clinical study using the nonsteroidalanti-inflammatory drug, indomethacin, indicated that indomethacinsignificantly slowed the progression of the disease (Neurology,43(8):1609 (1993)). In addition, a study examining age of onset among 50elderly twin pairs with onsets of AD separated by three or more years,suggested that anti-inflammatory drugs may prevent or delay the initialonset of AD symptoms (Neurology, 44:227 (1994)).

Over the years numerous therapies have been tested for the possiblebeneficial effects against EAE or MS disease but with mixed results(Cross et al., 1994; Ruuls et al., 1996). Though aminoguandine (AG) hasbeen described as a competitive inhibitor of iNOS and a suppressor ofits expression (Corbett and McDaniel, 1996; Joshi et al., 1996), to datefew compounds which inhibit iNOS are of potential therapeutic value havebeen identified. This deficiency is particularly troubling given thesignificant cellular damage which can arise as a result of iNOS-mediatednitric oxide toxicity, especially in chronic inflammatory diseasestates. There is a present need for therapeutic agents which willinhibit or even prevent cytotoxic concentrations of NO from occurring inindividuals suffering from diseases and conditions to which NO toxicityor an undesired production of proinflammatory cytokines is linked.

SUMMARY OF THE INVENTION

The invention generally provides methods of treating nitric oxide (NO)cytotoxicity comprising providing a biologically effective amount of aninducible nitric oxide synthase (iNOS) and/or proinflammatory cytokineinduction suppressor and/or inhibitor. The invention provides a solutionto the cytotoxicity induced or fostered by the presence of NO and/orproinflammatory cytokines which is observed in individuals sufferingfrom autoimmune or inflammatory diseases, including stroke,neurodegenerative diseases, demyelinating conditions (e.g., multiplesclerosis, experimental allergic encephalopathy,X-adrenoleukodystrophy), brain trauma, ischemia-reperfusion, Alzheimer'sdisease, aging, Landry-Guillain-Barre-Strohl syndrome, rheumatoidarthritis, endotoxic shock, myocardial infarction, tissue injury orHIV-mediated NO neurotoxicity.

The invention first provides a method for suppressing the induction ofinducible nitric oxide synthase and/or proinflammatory cytokines in acell comprising contacting said cell with an effective amount of atleast one induction suppressor and/or inhibitor of inducible nitricoxide synthase. Preferred cells throughout the various embodiments ofthe invention are lymphocytes, macrophages, endothelial cells,astrocytes, masengial cells, myocytes, Kuffer cells, epithelial cells,microglia, oligodendrocytes and neurons. Proinflammatory cytokines thatare preferred include TNF-α, IL-1β, IL-2, IL-6, IL-8 and IFN-γ. As usedherein certain embodiments “induction” may mean an increase in theoverall rate of gene transcription and/or translation. Induction mayalso mean that the rate of gene message or protein product destructionis decreased, producing a net increase in the amount of a message ortranslated protein. As used herein certain embodiments, the phrase“inhibition of nitric oxide cytotoxicity” denotes any measurabledecrease in the production of NO. Inhibition of nitric oxidecytotoxicity includes inhibition of iNOS activity, production of iNOSprotein, production or translation of iNOS mRNA, inhibition of LPS- orcytokine-induced NF-kβ activation in a cell. As used herein certainembodiments, “inhibitors” refers to such compounds or agents thatproduce any measurable decrease in the activity, production, orsecretion of a protein or biological compound, or the translation ofmRNA, in, or in the case of secretion, from, a cell. Proteins andbiological compounds that are specifically contemplated in the inventioninclude iNOS and proinflammatory cytokines. As used herein certainembodiments, a “enhancer” or “stimulator” refers to such compounds oragents that produce any measurable increase in the activity, production,or secretion of a protein or biological compound, or the translation ofmRNA, in, or in the case of secretion, from, a cell. As used hereincertain embodiments, an “inducer” refers to such compounds or agentsthat produce any measurable increase in the content, production,translation, or secretion of a protein or biological compound, or thetranslation of mRNA, in, or in the case of secretion, from, a cell. Asused herein certain embodiments, “a suppressor” refers to an agent orcompound that produces any measurable reduction in the induction of agene. Thus, a “suppressor” is a type of “inhibitor”, that acts reducethe net rate of transcription or translation of a target gene.

In preferred aspects of the invention, the induction suppressor and/orinhibitor of inducible nitric oxide synthase and/or proinflammatorycytokines may be selected from the group including, but not limited to,lovastatin, mevastatin, FPT inhibitor II, forskolin, rolipram,phenylacetate (NaPA), N-acetyl cysteine (NAC), pyrrolidinedithiocarbamate (PDTC), 4-phenylbutyrate (4PBA),5-aminoimmidazole-4-carboxamide ribonucleoside (AICAR), theophylline,papaverine, cAMP, 8-bromo-cAMP, (S)-cAMP, and salts, analogs, orderivatives thereof.

In some embodiments, the induction suppressor and/or inhibitor ofinducible nitric oxide synthase and/or proinflammatory cytokines may bean inhibitor of the Ras/Raf/MAP kinase pathway. In certain embodimentsthe induction suppressor and/or inhibitor of inducible nitric oxidesynthase and/or proinflammatory cytokines may be an inhibitor of NF-kB,such as for example an inhibitor of NF-kB activation, and/or asuppressor of its induction. In certain preferred embodiments theinhibitor of NF-kB activation includes, but is not limited to,lovastatin, NaPA, metastatin, 4-phenylbutyrate, FPT inhibitor II, AICARand salts, analogs, or derivatives thereof. In some embodiments, theinduction suppressor and/or inhibitor of inducible nitric oxide synthaseand/or proinflammatory cytokines may be an inhibitor of mevalonatesynthesis. In certain embodiments the inhibitor of mevalonate synthesismay be an inhibitor of the farnasylation of a protein. In certainpreferred embodiments the inhibitor of mevalonate synthesis may be aninhibitor of HMG-CoA reductase and/or suppressor of its induction,including but not limited to, lovastatin or AICAR and salts, analogs, orderivatives thereof. In certain preferred embodiments the inhibitor ofHMG-CoA reductase is a stimulator of AMP-activated protein kinase,including but not limited to, AICAR and salts, analogs, or derivativesthereof. In certain embodiments, the induction suppressor and/orinhibitor of inducible nitric oxide synthase and/or proinflammatorycytokines may be a stimulator of AMP-activated protein kinase. Incertain other preferred embodiments the inhibitor of inducible nitricoxide synthase and/or proinflammatory cytokines may be an inhibitor ofmevalonate pyrophosphate decarboxylase and/or suppressor of itsinduction, including but not limited to, phenylacetic acid,4-phenylbutyrate and salts, analogs, or derivatives thereof. In certainpreferred embodiments the inhibitor of mevalonate synthesis may belovastatin, mevastatin, NaPA, AICAR, 4-phenylbutyrate and salts,analogs, or derivatives thereof. In certain aspects embodiments theinhibitor of inducible nitric oxide synthase and/or proinflammatorycytokines is an inhibitor of farnesyl pyrophosphate. Preferredinhibitors of farnesyl pyrophosphate include, but are not limited to4-phenylbutyrate or NaPA.

In other embodiments the suppressor of inducible nitric oxide synthaseand/or proinflammatory cytokines is an antioxidant. In preferredembodiments the antioxidant may be, but is not limited to, N-acetylcysteine, PDTC, and salts, analogs, or derivatives thereof.

In certain other embodiments the inducible nitric oxide synthase and/orproinflammatory cytokines induction suppressor and/or inhibitor is anenhancer of intracellular cAMP, inhibitor of the Ras/Raf/MAP kinasepathway, and/or inhibitor of NF-kB, NF-kB activation and/or suppressorof NF-kB induction. In a preferred embodiment, the inhibitor of theRas/Raf/MAP kinase pathway includes, but is not limited to, AICAR andsalts, analogs, or derivatives thereof. The enhancer of intracellularcAMP may be an inhibitor of cAMP phosphodiesterase and/or suppressor ofits induction. In preferred aspects of the invention, the inhibitor ofcAMP phosphodiesterase may be, but is not limited to; rolipram andsalts, analogs, or derivatives thereof. In certain other aspects of theinvention, the induction suppressor and/or inhibitor of inducible nitricoxide synthase and/or proinflammatory cytokines is cAMP and salts,analogs, or derivatives thereof. Derivatives of cAMP include, but arenot limited to, 8-bromo-AMP or (S)-cAMP. In other aspects of theinvention, the enhancer of intracellular cAMP may be, but is not limitedto, forskolin, rolipram, 8-bromo-cAMP, theophylline, papaverine, cAMPand salts, analogs, or derivatives thereof. In certain embodiments, theinduction suppressor and/or inhibitor of inducible nitric oxide synthaseand/or proinflammatory cytokines may be a enhancer of protein kinase A.In other aspects of the invention, the enhancer of protein kinase A mayinclude, but is not limited to, forskolin, rolipram, 8-bromo-AMP,(S)-cAMP, cAMP and salts, analogs, or derivatives thereof. may be, butis not limited to, forskolin, rolipram, 8-bromo-cAMP, theophylline,papaverine, cAMP and salts, analogs, or derivatives thereof.

In yet another aspect of the invention, the induction suppressor and/orinhibitor of inducible nitric oxide synthase and/or proinflammatorycytokines may be a Ras farnesyl protein transferase inhibitor and/orinduction suppressor, an inhibitor of the farnasylation of Ras, and/oran activator of G-proteins. In a preferred embodiment, the Ras farnesylprotein transferase inhibitor and/or induction suppressor includes, butis not limited to, a FPT inhibitor and salts, analogs, or derivativesthereof. In a preferred embodiment, the inhibitor of the farnasylationof Ras, includes, but is not limited to, a FPT inhibitor II and salts,analogs, or derivatives thereof.

In one embodiment of the invention, the inducible nitric oxide synthaseand/or proinflammatory cytokines inhibitor and/or induction suppressoris selected from the group consisting of lovastatin, mevastatin, FPTinhibitor II, forskolin, rolipram, phenylacetate (NaPA), N-acetylcysteine (NAC), PDTC, 4-phenylbutyrate (4PBA),5-aminoimmidazole-4-carboxamide ribonucleoside (AICAR), theophylline,papaverine, cAMP, 8-bromo-cAMP, (S)-cAMP, and salts, analogs, orderivatives thereof. In a further embodiment of the invention,combinations of two or more inhibitors and/or induction suppressors arepreferred for use in the methods described herein.

A “salt” is understood herein certain embodiments to mean a compoundformed by the interaction of an acid and a base, the hydrogen atoms ofthe acid being replaced by the positive ion of the base. Salts, withinthe scope of this invention, include both the organic and inorganictypes and include, but are not limited to, the salts formed withammonia, organic amines, alkali metal hydroxides, alkali metalcarbonates, alkali metal bicarbonates, alkali metal hydrides, alkalimetal alkoxides, alkaline earth metal hydroxides, alkaline earth metalcarbonates, alkaline earth metal hydrides and alkaline earth metalalkoxides. Representative examples of bases that form such base saltsinclude ammonia, primary amines such as n-propylamine, n-butylamine,aniline, cyclohexylamine, benzylamine, p-toluidine, ethanolamine andglucamine; secondary amines such as diethylamine, diethanolamine,N-methylglucamine, N-methylaniline, morpholine, pyrrolidine andpiperidine; tertiary amines such as triethylamine, triethanolamine,N,N-dimethylaniline, N-ethylpiperidine and N-methylmorpholine;hydroxides such as sodium hydroxide; alkoxides such as sodium ethoxideand potassium methoxide; hydrides such as calcium hydride and sodiumhydride; and carbonates such as potassium carbonate and sodiumcarbonate. Preferred salts are those of sodium, potassium, ammonium,ethanolamine, diethanolamine and triethanolamine. Particularly preferredare the sodium salts.

As used herein, “derivatives” refers to chemically modified inhibitorsor stimulators that still retain the desired effects on property(s) ofiNOS or pro inflammatory gene, protein, and/or activity induction orsuppression. Derivatives may also retain other desired propertiesdescribed herein, such as suppressing the accumulation of very longchain fatty acids, defined herein as fatty acids with more than 22carbon atoms. Such derivatives may have the addition, removal, orsubstitution of one or more chemical moieties on the parent molecule.Such moieties may include, but are not limited to, an element such as ahydrogen or a halide, or a molecular group such as a methyl group. Sucha derivative may be prepared by any method known to those of skill inthe art. The properties of such derivatives may be assayed for theirdesired properties by any means described herein or known to those ofskill in the art.

As used herein, “analogs” include structural equivalents or mimetics,described further in the detailed description.

In administering the inducible nitric oxide synthase and/orproinflammatory cytokines inhibitors and/or induction suppressors to amammal, preferably a human, pig, cats, dogs, rodent, or cattle includingbut not limited to, sheep, goats and cows, the inhibitor is formulatedin a pharmaceutically acceptable vehicle. The induction suppressorand/or inhibitor may be administered to a patient in a dose therapeuticto treat a diseases, conditions and disorders where there is anadvantage in inhibiting the nitric oxide synthase enzyme and/or theproduction of proinflammatory cytokines.

A “patient”, as used herein, may be an animal. Preferred animals aremammals, including but not limited to humans, pigs, cats, dogs, rodents,or cattle including but not limited to, sheep, goats and cows. Preferredpatients are humans.

The induction suppressors, also known as “suppressing agents”, and/orinhibitors of iNOS and/or proinflamrnatory cytokines, in pure form or ina pharmaceutically acceptable carrier, will find benefit in treatingconditions and disorders, described below, where there is an advantagein inhibiting and/or suppression the induction of proinflammatorycytokines and/or the inducible isoform of nitric oxide synthase enzyme.These induction suppressors and/or inhibitors may also be used to treatconditions and disorders created, induced, enhanced and/or aggravated bythe contact of a cell with bacterial endotoxin (LPS).

For example, the suppressing agents and/or inhibitors may be used totreat circulatory shock including its various aspects such as vascularand myocardial dysfunction, metabolic failure including the inhibitionof mitochondrial enzymes and cytochrome P450-mediated drug metabolism,and multiple organ dysfunction syndrome including adult respiratorydistress syndrome. Hypotension and/or circulatory shock may be a resultof gram-negative and gram positive sepsis (a.k.a. septic shock), toxicshock, trauma, hemorrhage, burn injury, anaphylaxis, cytokineimmunotherapy, liver failure, kidney failure or systemic inflammatoryresponse syndrome. Suppressing agents and/or inhibitors also may bebeneficial for patients receiving therapy, including cancer therapy,with cytokines such as TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ, ortherapy with cytokine-inducing agents, or as an adjuvant to short termimmunosuppression in transplant therapy. In addition, the suppressingagents and/or inhibitors may be useful to inhibit NO synthesis inpatients suffering from inflammatory conditions in which an excess of NOcontributes to the pathophysiology of the condition, such as adultrespiratory distress syndrome (ARDS) and myocarditis, for example.

There is also evidence that an NO synthase enzyme and/or proinflammatorycytokines may be involved in the pathophysiology of autoimmune and/orinflammatory conditions such as arthritis, rheumatoid arthritis andsystemic lupus erythematosus (SLE) and in insulin-dependent diabetes,mellitus type 1 diabetes, and therefore, the suppressing agents mayprove helpful in treating these conditions.

Furthermore, it is now clear that there are a number of additionalinflammatory and noninflammatory diseases and/conditions that areassociated with NO overproduction Examples of such physiologicaldisorders include: inflammatory bowel diseases such as ileitis,ulcerative colitis and Crohn's disease; inflammatory lung disorders suchas asthma, bronchitis, oxidant-induced lung injury and chronicobstructive airway disease; inflammatory disorders of the eye includingcorneal dystrophy, ocular hypertension, trachoma, onchocerciasis,retinitis, uveitis, sympathetic ophthalmitis and endophthalmitis;chronic inflammatory disorders of the gum including periodontitis;chronic inflammatory disorders of the joints including arthritis, septicarthritis and osteoarthritis, tuberculosis, leprosy, glomerulonephritissarcoid, and nephrosis; disorders of the skin includingsclerodermatitis, sunburn, psoriasis and eczema; inflammatory diseasesof the central nervous system, including amyotrophic lateral sclerosis,chronic demyelinating diseases such as multiple sclerosis, dementiaincluding AIDS-related neurodegeneration and Alzheimer's disease,encephalomyelitis and viral or autoimmune encephalitis; autoimmunediseases including immune-complex vasculitis, systemic lupus anderythematosis; and disease of the heart including ischemic heartdisease, heart failure and cardiomyopathy. Additional disease that maybenefit from the use of suppressing agents include adrenalinsufficiency; hypercholesterolemia; atherosclerosis; bone diseaseassociated with increased bone resorption, e.g., osteoporosis,pre-eclampsia, eclampsia, uremic complications; chronic liver failure,noninflammatory diseases of the central nervous system (CNS) includingstroke and cerebral ischemia; and other disorders associated withinflammation and undesirable production of nitric oxide and/orproinflamatory cytokines such as cystic fibrosis, tuberculosis,cachexia, ischeimia/reperfusion, hemodialysis related conditions,glomerulonephritis, restenosis, inflammatory sequelae of viralinfections, hypoxia, hyperbaric oxygen convulsions and toxicity,dementia, Sydenham's chorea, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis, epilepsy,Korsakoff's disease, imbecility related to cerebral vessel disorder, NOmediated cerebral trauma and related sequelae, ischemic brain edema(stroke), pain, migraine, emesis, immune complex disease, asimmunosuppressive agents, acute allograft rejection, infections causedby invasive microorganisms which produce NO and for preventing orreversing tolerance to opiates and diazepines, aging, and various formsof cancer. All these nitric oxide and/or proinflammatory cytokine and/orendotoxin induced, mediated, enhanced, and/or aggravated diseases anddisorders are contemplated as being treatable in a cell by contactingthe cell with at least one suppressing agent and/or inhibitor of iNOSand/or proinflammatory cytokines. A patient with may also be treated byadministering at least one suppressing agent and/or inhibitor of iNOSand/or proinflammatory cytokines. When administered to a patient, the atleast one suppressing agent and/or inhibitor is formulated in apharmaceutically acceptable vehicle.

In another aspect the present invention provides a method ofidentifying, or screening for, a candidate inducible nitric oxidesynthase and/or proinflammatory cytokines inhibitor and/or inductionsuppressor, comprising preparing a cell capable of producing induciblenitric oxide synthase and/or proinflammatory cytokines activity andtesting the candidate inhibitor and/or induction suppressor for theability to inhibit the inducible nitric oxide synthase and/orproinflammatory cytokines activity, wherein the inhibition is indicativeof a candidate inducible nitric oxide synthase and/or proinflammatorycytokines inhibitor and/or induction suppressor. These candidateinhibitor and/or induction suppressor are known herein as “candidatesubstances”. A further aspect of this method is to identify an iNOSspecific inhibitor and/or induction suppressor that does not inhibit orsuppress one or more proinflammatory cytokines. Another aspect of thisinvention is to identify an inhibitor and/or induction suppressor thatdoes not inhibit or suppress iNOS, but does inhibit or suppress one ormore proinflammatory cytokines.

This method of identifying a candidate inducible nitric oxide synthaseand/or proinflammatory cytokines induction suppressor and/or inhibitorcomprising the steps of a) obtaining a cell comprising at least thecapability of producing inducible nitric oxide synthase and/orproinflammatory cytokines activity; b) obtaining a candidate induciblenitric oxide synthase and/or proinflammatory cytokines inductionsuppressor and/or inhibitor; c) contacting the cell with the candidateinducible nitric oxide synthase and/or proinflammatory cytokinesinduction suppressor and/or inhibitor under conditions normallyinducing, enhancing, and/or stimulating iNOS and/or proinflammatorycytokines; and d) determining the ability of the candidate induciblenitric oxide synthase and/or proinflammatory cytokines inductionsuppressor and/or inhibitor to inhibit the formation of nitric oxide inthe presence of inducible nitric oxide synthase, wherein the inhibitionof the formation of nitric oxide in the presence of inducible nitricoxide synthase is indicative of a candidate inducible nitric oxidesynthase induction suppressor and/or inhibitor. In an aspect of theinvention, decreased content or production of at least oneproinflammatory cytokine by a cell is indicative of a candidateproinflammatory cytokine induction suppressor. In another aspect of theinvention, decreased bioactivity of at least one proinflammatorycytokine is indicative of a candidate proinflammatory cytokine inhibitorand/or induction suppressor. In further aspects of this method, aninduction suppressor and/or inhibitor is further identified by detectingthe amount of iNOS and/or proinflammatory cytokine mRNA message and/orprotein content and/or biological activity. In additional aspects of theinvention, an induction suppressor and/or inhibitor is furtheridentified by comparing the amount of iNOS and/or proinflammatorycytokine mRNA message and/or protein content and/or biological activityto another cell under conditions normally inducing, enhancing, and/orstimulating iNOS and/or proinflammatory cytokines in the absence of thecandidate inhibitor and/or induction suppressor. The preferredconditions inducing, enhancing, and/or stimulating iNOS and/orproinflammatory cytokines is contacting a cell with endotoxin and/or atleast one cytokine and/or at least one inducer or stimulator of at leastone proinflammatory cytokine. Preferred cytokines are proinflammatorycytokines.

In preferred embodiments, a candidate induction suppressor and/orinhibitor of inducible nitric oxide synthase and/or proinflammatorycytokines is selected from agents that have certain traits or modes ofaction common to those of the suppressors and/or inhibitors identifiedherein. Preferred candidate substances would either inhibit theRas/Raf/MAP kinase pathway, inhibit and/or suppress the induction and/oractivation of NF-kB, inhibit mevalonate synthesis, be an enhancer ofprotein kinase A, and/or inhibit the farnasylation of proteins,including but not limited to Ras. In certain embodiments the inhibitorof mevalonate synthesis may be an inhibitor of HMG-CoA reductase orsuppressor of its induction. In certain aspects the inhibitor of HMG-CoAreductase is a stimulator of AMP-activated protein kinase. In certainother embodiments the inhibitor of inducible nitric oxide synthaseand/or proinflammatory cytokines may be an inhibitor of mevalonatepyrophosphate decarboxylase or suppressor of its induction. In otherembodiments the candidate substance is an antioxidant. In otherembodiments the candidate substance is an enhancer of intracellularcAMP. The enhancer of intracellular cAMP may be an inhibitor of cAMPphosphodiesterase and/or suppressor of its induction. In otherembodiments the candidate substance is a farnesyl protein transferaseinhibitor and/or induction suppressor.

Proinflammatory cytokine and/or iNOS RNA message, protein content, oractivity can be detected by any method described herein or known tothose of skill in the art (see for example, Sambrook et al., 1989), andinclude but are not limited to Northern analysis of iNOS and/orinflammatory cytokine message, PCR™ amplification of target message,immunodetection techniques including Western analysis of iNOS and/orproinflammatory cytokine content or production, and chemical orbiological activity assays for iNOS or cytokine activity.

Candidate inhibitors and/or induction suppressors identified by themethod of the invention are preferably purified. When administered to amammal, the purified candidate inducible nitric oxide synthase inhibitorand/or induction suppressor is formulated in a pharmaceuticallyacceptable vehicle.

In another preferred embodiment, the invention provides a method ofinhibiting nitric oxide cytotoxicity comprising contacting a cellcapable of producing nitric oxide with a biologically effective amountof at least one inducible nitric oxide synthase induction suppressorand/or inhibitor identified by the screening assay of the invention. Inpreferred embodiments, the cell is in a patient.

In another preferred embodiment, the invention provides a method ofinhibiting proinflammatory cytokine or endotoxin treated, induced oraggravated conditions and disorders, where there is an advantage ininhibiting and/or suppression the induction of proinflammatorycytokines. In certain embodiments, the method comprises contacting acell with a biologically effective amount of at least one inductionsuppressor and/or inhibitor of: at least one proinflammatory cytokineand/or iNOS. In certain aspects of the invention, the at least oneinduction suppressor and/or inhibitor is identified by the screeningassay of the invention. In preferred embodiments, the cell is in apatient.

The invention also provides a method of suppressing the accumulation ofvery long chain fatty acids in a cell, by contacting the cell with abiologically effective amount of at least induction suppressor and/orinhibitor of: inducible nitric oxide synthase and/or at least oneproinflammatory cytokine. In certain aspects of the invention, the atleast one induction suppressor and/or inhibitor is identified by thescreening assay of the invention. In preferred embodiments, the cell isin a patient. Such methods have use in inflammatory conditionsincluding, but not limited to, demylenating diseases or neural trauma,and particularly in treating patients with X-ALD. In certain aspects ofthe invention, lignoceric acid β-oxidation is stimulated. In otheraspects of the invention, the ratios of C_(26:0)/C_(22:0) orC_(24:0)/C_(22:0) fatty acids are lowered.

The invention provides a method of treating a nitric oxide and/orcytokine mediated disorder in a cell, by contacting the cell with abiologically effective amount of at least one induction suppressorand/or inhibitor of: inducible nitric oxide synthase and/or at least oneproinflammatory cytokine. In certain aspects of the invention, the atleast one induction suppressor and/or inhibitor is identified by thescreening assay of the invention. In preferred embodiments, the cell isin a patient. In preferred aspects, the disorder is X-ALD, multiplesclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, lupus,septic shock, stroke, ischemia/reperfusion, rheumatoid arthritis,osteoarthritis or aging. In other preferred aspects, the nitric oxide orcytokine mediated disorder is myelinolytic inflammation, a demyelinatingcondition or an inflammatory demyelinating disease, or aneuroinflammatory disease. The inflammatory disease is preferably X-ALD,multiple sclerosis, Landry-Guillain-Barre-Strohl syndrome, Alzheimer'sdisease and/or aging.

In another preferred embodiment, the invention provides a method oftreating septic shock comprising contacting a cell capable of producingexcess nitric oxide and/or at least one proinflammatory cytokine underconditions of septic shock with a biologically effective amount of aninducible nitric oxide synthase and/or proinflammatory cytokineinduction suppressor and/or inhibitor. In certain aspects of theinvention, the induction suppressor and/or inhibitor is identified bythe screening assay of the invention. In preferred aspects of theinvention, the cell is in a patient. Methods of treating septic shockwith inhibitors of nitric oxide synthase activity are described in U.S.Pat. Nos. 5,028,627 and 5,296,466, each incorporated herein by referencein entirety.

The present invention is further directed to methods for inducing orsuppressing apoptosis in the cells and/or tissues of individualssuffering from degenerative disorders characterized by inappropriatecell proliferation or inappropriate cell death, or in some cases, both.The method comprises contacting a cell capable of producing excessnitric oxide under conditions of degenerative disorders with abiologically effective amount of an inducible nitric oxide synthaseand/or proinflammatory cytokines induction suppressor and/or inhibitor.In preferred aspects of the invention, the cell is in a patient. Incertain aspects of the invention, the cytokines induction suppressorand/or inhibitor identified by the screening assay of the invention.Inappropriate cell proliferation will include the statisticallysignificant increase in cell number as compared to the proliferation ofthat particular cell type in the normal population. Also included aredisorders whereby a cell is present and/or persists in an inappropriatelocation, e.g., the presence of fibroblasts in lung tissue after acutelung injury, and cancer cells which exhibit the properties of invasionand metastasis and are highly anaplastic. Such cells include but are notlimited to, cancer cells including, for example, tumor cells.Inappropriate cell death will include a statistically significantdecrease in cell number as compared to the presence of that particularcell type in the normal population. Such underrepresentation may be dueto a particular degenerative disorder, including, for example, viralinfections such as AIDS (HIV), which results in the inappropriate deathof T-cells, and autoimmune diseases which are characterized byinappropriate cell death. Autoimmune diseases are disorders caused by animmune response directed against self antigens. Such diseases arecharacterized by the presence of circulating autoantibodies orcell-mediated immunity against autoantigens in conjunction withinflammatory lesions caused by immunologically competent cells or immunecomplexes in tissues containing the autoantigens. Such diseases includesystemic lupus erythematosus (SLE), rheumatoid arthritis. Standardreference works setting forth the heneral principles of immunologyinclude Stites and Terr, 1991 and Abbas et al., 1991.

The invention particularly relates to the use of at least one iNOSand/or pro-inflammatory cytokine induction suppressor and/or inhibitors,preferably reductants such as NAC or other thiol compounds to reduceNO-mediated cytotoxicity as well as ceramide-mediated apoptosis inneuroinflammatory diseases and degenerative disorders. Suppressingagents in this class would be particularly preferred in treatingdiseases characterized by excessive or inappropriate cell death,including, for example, neuro-degenerative diseases and injury resultingfrom ischemia. Degenerative disorders characterized by inappropriatecell proliferation include, for example, inflammatory conditions,cancer, including lymphomas, such as prostate hyperplasia, genotypictumors, etc. Degenerative disorders characterized by inappropriate celldeath include, for example, autoimmune diseases, acquiredimmunodeficiency disease (AIDS), cell death due to radiation therapy orchemotherapy, neurodegenerative diseases, such as Alzheimer's disease,Parkinson's disease, Landry-Guillain-Barre-Strohl syndrome, multiplesclerosis, etc. In certain aspects of the invention, the at least oneinduction suppressor and/or inhibitor is identified by the screeningassay of the invention.

The invention further provides a method for enhancing the production ofan inducible nitric oxide synthatase or a proinflammatory cytokine in acell comprising providing a biologically effective amount of a induciblenitric oxide synthatase and/or proinflammatory cytokine stimulator. Incertain aspects of the invention, the at least one induction stimulatorand/or enhancer is identified by the screening assay of the invention. Astimulator in this aspect of the invention is preferably an inductionstimulator. Preferred stimulators include a PKA inhibitor or enhancer ofintracellular cAMP. PKA inhibitors may include, but are not limited to,H-89, myristoylated PKI, (R)-cAMP and salts, analogs, or derivativesthereof. The enhancers of intracellular cAMP may also be selected fromthe group comprising forskolin, 8-bromo-cAMP and rolipram. In otherpreferred aspects of the invention, the enhancer of intracellular cAMPis an inhibitor of cAMP phosphodiesterase. A preferred inhibitor of cAMPphosphodiesterase is rolipram. In other aspects of this method, abiologically effective amount of LPS and/or one or more proinflammatorycytokine is administered to stimulate iNOS and/or proinflammatorycytokines' induction or activity. Preferred proinflammatory cytokinethat are administered include TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ.

Following long-standing patent law convention, the words “a” and “an,”as used in this specification, including the claims, denotes “one ormore.” Specifically, the use of “comprising,” “having,” or other openlanguage in claims that claim a combination or method employing “anobject,” denotes that “one or more of the object” may be employed in theclaimed method or combination.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Forskolin inhibits LPS-induced NO production and iNOS activityin a dose-dependent manner in rat primary astrocytes. Cells incubated inserum-free DMEM/F-12 received different concentrations of forskolin 15min before the addition of 1.0 μg/ml LPS. The production of nitrite insupernatants (◯) and activities of iNOS in cell homogenates (●) weremeasured after 24 h of incubation as described in Example 6. PKAactivity was measured in cell homogenates (□) after 1 h of incubation.Nitrite production in supernatants (32.3±3.6 nmol/mg/24 h), and iNOSactivity in homogenates (48.7±3.9 pmol/min/mg) found in cells stimulatedwith only LPS are considered as 100%. However, PKA activity found inextracts from cells stimulated with an optimal concentration offorskolin (74.4±9.4 pmol/min/mg) is considered as 100%. Values are meanof duplicate samples.

FIG. 2. Activation of PKA correlates with the stimulation of β-oxidationand inhibition of fatty acid chain elongation in cultured skinfibroblasts of X-ALD. Cells were treated for 72 h serum-containing DMEMwith the listed reagents; β-oxidation of lignoceric acid (FIG. 2A),fatty acid chain elongation (FIG. 2B) and PKA (FIG. 2C) activities weremeasured as described in Example 4. Media was replaced after every 24 hwith the addition of fresh reagents. Concentrations of reagents were:forskolin, 4 μM; 8-Br-cAMP, 50 μM; rolipram, 10 μM; H-89, 1 μM;myristoylated PKI, 0.2 μM. Data are mean±S.D. of three differentexperiments.

FIG. 3. Time-dependent effect of forskolin on the ratios of VLCFA(C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0)) and β-oxidation of lignocericacid in cultured skin fibroblasts of X-ALD. Cells were incubated inserum-containing DMEM with 4 μM forskolin for different days, and theratios of C_(26:0)/C_(22:0) (FIG. 3A) and C_(24:0)/C_(22:0) (FIG. 3B),and β-oxidation of lignoceric acid (FIG. 3C) were measured as describedin Example 4 (O, experiment 1; O, experiment 2).

FIG. 4. Modulators of PKA modulate the induction of TNF-α and IL-1β inrat primary astrocytes. Cells preincubated with the listed reagents for15 min in serum-free condition was stimulated with 1.0 μg/ml of LPS.After 24 h of incubation, concentrations of TNF-α (FIG. 4A) and IL-1β(FIG. 4B) were measured in supernatants as described in the methodssection. After 1 h of incubation, activity of PKA (FIG. 4C) was measuredin cell extracts as described in Example 4. TNF-α and IL-1β areexpressed as ng/24 h/mg protein. Data are expressed as the mean±S.D. ofthree different experiments. Concentrations of reagents were: forskolin,10 μM; 8-Br-cAMP, 100 μM; rolipram, 20 μM; H-89, 2 μM; myristoylatedPKI, 0.4 μM. Data are mean±S.D. of three different experiments.

FIG. 5. Effect of okadaic acid on iNOS promoter-derived CAT activity inrat primary astrocytes and macrophages. Astrocytes (FIG. 5A) macrophages(FIG. 5B) were transfected with the construct containing the iNOSpromoter fused to the CAT gene using lipofectamine. Twenty four hourafter transfection, cells received okadaic acid with or without 1.0μg/ml of LPS and after 14 h of stimulation, CAT activity was measured.Data are mean±S.D. of three different experiments.

FIG. 6. Inhibition of TNF-α-induced degradation of sphingomyelin toceramide by NAC and PDTC in rat primary astrocytes. Cells preincubatedwith either 10 mM NAC or 100 μM PDTC for 1 h in serum-free DMEM/F-12received TNF-α (50 ng/ml). At different time intervals, cells werewashed with HBSS and scrapped off Lipids were extracted, and levels ofceramide (FIG. 6A) and sphingomyelin (FIG. 6B) were measured asdescribed in Example 7. Ceramide levels are expressed as -fold changeover the level at 0 min. Results are mean±S.D. of three differentstudies.

FIG. 7. NAC and PDTC inhibit IL-1β-mediated degradation of sphingomyelinto ceramide in rat primary astrocytes. Cells preincubated with either 10mM NAC or 100 μM PDTC for 1 h in serum-free DMEM/F-12 received IL-1β (50ng/ml). At different time intervals, cells were washed with HBSS andscrapped off Lipids were extracted, and levels of ceramide (FIG. 7A) andsphingomyelin (FIG. 7B) were measured as described in Example 7.Ceramide levels are expressed as -fold change over the level at 0 min.Results are mean±S.D. of three different studies.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention discloses the novel uses of compounds which inhibit theinduction of iNOS and/or proinflammatory cytokines and the production ofNO and/or proinflammatory cytokines by cells, including lymphocytes,macrophages, endothelial cells, astrocytes and microglia in response toinflammatory cytokines for the therapeutic treatment of diseaseaffecting the vascular and nervous systems. The therapeutic usesdescribed herein utilizing these compounds provide protection against NOtoxicity to including lymphocytes, macrophages, endothelial cells,astrocytes microglia, oligodendrocytes and neurons in neuroinflammatorydisease, stroke, ischemia-reperfusion and tissue injury and HIV-mediatedNO neurotoxicity for which there is no effective treatment presentlyavailable.

The present disclosure further describes the discovery of a novel roleof the mevalonate pathway in controlling the expression of iNOS anddifferent cytokines in lymphocytes, macrophages, endothelial cells,astrocytes and microglia. This discovery provides the basis for novelscreening assays of previously unknown inhibitors of iNOS and theproduction of NO. An understanding of the cellular mechanisms involvedin the induction of iNOS and cytokines allows identification of noveltargets for the therapeutic intervention of NO-mediated, proinflammatorycytokine and/or endotoxin-mediated pathophysiology in inflammatorydiseases.

The inventor demonstrates herein that LPS- and cytokine-inducedproduction of NO can be blocked by antioxidants. Therefore maintenanceof the thiol/oxidant balance appears to be crucial for protectionagainst proinflammatory cytokine production and, at least, in NOcytotoxicity. The inventor has discovered that the use of reductants,such as N-acetyl cysteine (NAC) or other thiol compounds, is beneficialin restoring cellular redox and in inhibiting the production ofproinflammatory cytokines and in reducing cytotoxic levels of NO.N-acetyl cysteine blocks the induction of TNF-α and iNOS and is anontoxic drug that enters the cell readily and serves both as ascavenger of reactive oxygen species and a precursor of glutathione, themajor intracellular thiol (Smilkstein et al., 1988; Aruoma et al.,1989). Therefore, the use of reductants such as NAC or other thiolcompounds, may be beneficial in restoring cellular redox and ininhibition of production of proinflammatory cytokines and in reducingcytotoxic levels of NO.

The inventor investigated the cellular regulation of the induction ofiNOS and cytokines by lovastatin and NaPA in rat primary lymphocytes,macrophages, endothelial cells, astrocytes and microglia. Thisinvestigation disclosed the first evidence that the induction ofinducible nitric oxide synthase (iNOS) and cytokine (for example TNF-α,IL-1β and IL-6) gene expression are uniquely sensitive to the drugslovastatin and the sodium salt of phenylacetic acid (NaPA) inastrocytes, glial cells and macrophages. The reversal oflovastatin-mediated inhibition of iNOS induction by mevalonate and FPP,and reversal of the inhibitory effect of NaPA by FPP, and inhibition ofRas farnesyl protein transferase by an inhibitor (FPT inhibitor II)demonstrated that the farnesylation reaction is a key step in theregulation of LPS-mediated induction of iNOS and production of NO andcytokines.

The inventor have discovered that lovastatin and NaPA, alone or incombination represent therapeutic agents directed against cytokine- andnitric oxide-mediated brain disorders, particularly in stroke, trauma,Alzheimer's Disease and in demyelinating conditions such as multiplesclerosis and X-adrenoleukodystrophy (X-ALD).

The inventor's results demonstrate that the inhibition iNOS expressionby lovastatin, NaPA and FPT inhibitor II may be due to the inhibition ofNF-kβ activation. Previous studies of Law et al. (1992) demonstratingthe inhibition of NF-kβ activation by mevinolin and5′-methylthioadenosine indicated a role of protein farnesylation andcarboxyl methylation reactions in the activation of NF-kβ. The Rasprotooncogene proteins function by binding to cytoplasmic surface ofplasma membrane. Since mevalonate availability regulates thepost-translational isoprenylation of many intracellular signalingproteins including Ras p21 (Goldstein et al., 1990), the observedinhibition of NF-kβ activation and induction of iNOS by lovastatin andNaPA appears to be due to decreased, or a lack of, isoprenylation of Rasthat in turn leads to the lack of or abnormal signal transmission fromreceptor tyrosine kinase to Ras/Raf/MAP kinase cascade, activation ofNF-kβ and induction of iNOS.

The inventor has also investigated the effect of other antioxidants onthe induction of NO by LPS and/or cytokine-stimulated macrophages, C6glioma cell lymphocytes, endothelial cells, astrocytes and microglia.These results clearly show that antioxidants (N-acetyl cysteine (NAC)and pyrrolidine dithiocarbamate (PDTC)) inhibit the LPS- andcytokine-induced production of NO, iNOS activity, production of iNOSprotein and iNOS mRNA indicating a role of reactive oxygen species(e.g., H₂O₂, O₂ and OH) in iNOS induction. Superoxide (O₂ ⁻) andhydroxyl radical (OH) are reported to be involved in the production ofNO in brain cerebellum (Mittal, 1993) where the hydroxyl radical wasindicated to hydroxylate L-arginine during its conversion to citrullineand NO (Mittal, 1993). The inventor has discovered through theinhibition of iNOS activity and induction of iNOS protein and mRNA inLPS- and cytokine-activated macrophages by NAC that reactive oxygenspecies (ROS) modulate the intracellular signal pathways for theinduction of iNOS biogenesis.

Several lines of evidence disclosed herein clearly support theconclusion that inhibitors of HMG-CoA reductase (for example, lovastatinor mevastatin) and mevalonate pyrophosphate decarboxylase (NaPA) have aninhibitory effect on the induction of inflammatory mediators (iNOS,TNF-α, IL-1β and IL-6) in rat astrocytes, microglia and macrophagesdemonstrating the involvement of mevalonate metabolite(s), farnesylpyrophosphate, in the induction of inflammatory mediators. Thisconclusion was based on the following observations. First, theLPS-induced expression of iNOS, TNF-α, IL-1β and IL-6, and activation ofNF-kβ was inhibited by lovastatin and NaPA. Second, inhibitory effectsof lovastatin and NaPA on LPS-mediated induction of iNOS and cytokineswas not reversed by cholesterol and ubiquinone, end products of themevalonate pathway, indicating that this inhibitory effect of lovastatinwas not due to depletion of end products of mevalonate pathway. Third,the reversal of inhibitory effects of lovastatin by mevalonate and FPPand the reversal of inhibitory effects of NaPA by FPP, but not bymevalonate, indicates a role of farnesylation in LPS-mediated inductionof iNOS. Fourth, the inhibition of LPS-induced activation of NF-kβ andinduction of iNOS by FPT inhibitor II, an inhibitor of Ras farnesylprotein transferase, demonstrates that farnesylation of Ras is requiredfor signal transduction in the LPS-induced expression of iNOS. Since theiNOS, TNF-α, IL-1β and IL-6 have been implicated in the pathogenesis ofdemyelinating and neurodegenerative diseases (Mitrovic et al., 1994; Boet al, 1994; Merrill et al., 1993), these results provide an importantmechanism whereby inhibitors of HMG-CoA reductase and mevalonatepyrophosphate decarboxylase can ameliorate neural injury.

Therapy For X-Adreno Leukodystrophy

Since X-ALD is a metabolic disorder of the very long chain fatty acids(VLCFA) that eventually leads to an inflammatory bilateral demyelinationwith marked activation of microglia and astrocytes and accumulation ofproinflammatory cytokines (TNF-α and IL-1β) and extracellular matrixproteins (Powers et al., 1992; McGuinness et al., 1995), the inventordeveloped a therapy that should normalize the VLCFA and inhibit theinduction of proinflammatory cytokines by astrocytes and microglia.Example 4 described herein demonstrates that the compounds that increasethe intracellular levels of cAMP and the activity of protein kinase A(PKA) normalize the levels of VLCFA possibly by increasing theperoxisomal activity for β-oxidation of VLCFA. Moreover, the samecompounds also inhibit the induction of TNF-α and IL-1β inlipopolysaccharide (LPS) stimulated astrocytes and microglia. Theseobservations demonstrate the therapeutic potential of compounds thatincrease the activity of PKA in correction of the metabolic defect andinhibition of the neuroinflammatory disease process in X-ALD.

The inventor provides evidence that in X-ALD cultured skin fibroblasts,up regulation of PKA activity increased the β-oxidation of lignocericacid, decreased the chain elongation of fatty acids and lowered cellularcontent of VLCFA to the normal level, despite the status (mutation ordeletion) of the ALD gene. The detailed mechanism leading to thenormalization of VLCFA in X-ALD is not known at the present, but islikely to involve cAMP-dependent protein kinase A. This conclusion isbased on the following observations. First, cAMP analogs and rolipram,an inhibitor of cAMP phosphodiesterase, stimulated transport andβ-oxidation of lignoceric acid and decreased the chain elongation offatty acids in X-ALD as well as control skin fibroblasts whereas H-89and myristoylated PKI, specific inhibitors of PKA, inhibited transportand β-oxidation of lignoceric acid, stimulated chain elongation of fattyacids and blocked the observed effects in normalization of VLCFA by cAMPanalogs. Second, a long-term treatment of fibroblasts of X-ALD with cAMPanalogs and rolipram although had no effect on protein and mRNA forX-ALD gene but lowered the accumulation of VLCFA to the control levelthat is also blocked by inhibitors of PKA. These results clearlyindicate that increasing cAMP level in fibroblasts of X-ALD normalizesthe VLCFA pathogen by a mechanism that is dependent on the activity ofPKA but independent of the involvement of the ALD gene product.

Previous studies (Singh et al., 1984; Hashmi et al., 1986; Lageweg etal., 1991; Lazo et al., 1988; Lazo et al., 1989) have shown that VLCFA(lignoceric and cerotic acids) are preferentially β-oxidized inperoxisomes. The increased transport of lignoceric acid intocAMP-treated cells indicates that the observed increase in β-oxidationof lignoceric acid is due to higher availability of lignoceric acid inthese cells. However, the increase in β-oxidation of lignoceric acid incell-free extracts or permealized X-ALD cells, or cell homogenatesdemonstrate that normalization of VLCFA is due to increased activity offatty acid β-oxidation system. In the cell, fatty acids are β-oxidizedin mitochondria and peroxisomes (Singh, 1997). The lack of effect ofetomoxir, an inhibitor of mitochondrial carnitine palmitoyltransferase-I (Mannaerts et al., 1979), on the cAMP-stimulated oxidationindicates that the higher lignoceric acid oxidation activity observed incAMP-stimulated cells was due to increase in the activity of peroxisomalβ-oxidation system. These observations provide the first evidence thatperoxisomal β-oxidation of fatty acids is regulated by intracellularsecond messenger (cAMP).

The pathogenetic mechanism of X-ALD is poorly understood. The constant“hallmark” of X-ALD is an excessive accumulation of VLCFA withsubsequent involvement of CNS with induction of proinflammatorycytokines (TNF-α and IL-1β) and extracellular matrix proteins byreactive astrocytes and microglia and demyelination/inflammatorydysmyelination and loss of oligodendrocytes (Powers et al., 1992;McGuinness et al., 1995; Powers, 1995). The documentation ofimmunoreactive TNF-α and IL-1β in astrocytes and microglia of X-ALDbrain indicated the involvement of these cytokines in immunopathology ofX-ALD and aligned X-ALD with multiple sclerosis (MS), the most commonimmune-mediated demyelinating disease of the CNS in man. However, apartfrom traditionally higher expression of cytokines by microglia than inastrocytes of MS and other neurodegenerative disorders, the expressionof TNF-α and IL-1β is more prominent in astrocytes than microglia ofX-ALD brain (Powers et al., 1992).

At present it is not known how the inherited metabolic abnormality ofaccumulation of VLCFA subsequently triggers a neuroinflammatory responsein X-ALD brain. Since the metabolic defect appears prior to thedetection of neuroinflammatory disease, the assumption is that theseVLCFA, by themselves or as a constituent of complex lipid, act as atrigger for the inflammatory response that in turn becomes the basis forthe observed demyelination and loss of oligodendrocytes in X-ALD. Thedata presented here indicate that cAMP may also inhibit the induction ofproinflammatory cytokines in reactive astrocytes and microglia. Thetreatments of rat brain primary astrocytes or microglia with forskolinor rolipram inhibit the LPS-induced induction of TNF-α and IL-1β.

Previously it has been shown that cAMP derivatives and rolipram inhibitthe cytokine-induced expression of inducible nitric oxide synthase andproduction of NO in astrocytes. The inventor's studies indicate thatproinflammatory cytokines down regulate the peroxisomal function in themetabolism of VLCFA thereby aggravating the inherited metabolicabnormality by accumulating 4-times higher VLCFA and around the plaquethan in normal looking X-ALD brain and these alterations byproinflammatory cytokines are mediated by NO toxicity (Khan et al.,1997). The inhibition of induction of cytokines as well as induction ofiNOS by compounds that increase the activity of PKA (e.g., cAMP androlipram) in astrocytes and microglia indicate that these compoundsshould be beneficial in terms of blocking the induction ofproinflammatory cytokines in X-ALD

These results provide the basis of a therapy to normalize the metabolicabnormality and block the neuroinflammatory process by inhibiting theinduction of proinflammatory cytokines. The studies described in Example4 clearly demonstrate that the compounds (e.g. forskolin, 8-Br-cAMP,rolipram) that increase cAMP and activate PKA meet both of theseconditions. Moreover, recent reports showing the prevention ofprogression of autoimmune encephalomyelitis in mice (Sommer et al.,1995) as well as in marmoset by rolipram indicate that rolipram doescross the blood brain barrier and inhibit the cytokine-inducedneuropathologies in these animal models.

The studies described in Example 5 demonstrate that lovastatin andsodium phenylacetate (NaPA), inhibitors of mevalonate pathway, normalizethe levels of VLCFA in skin fibroblasts of X-ALD by increasing theperoxisomal activity for β-oxidation of VLCFA. In light of the fact thatthese compounds also inhibit the induction of proinflammatory cytokinesand nitric oxide synthase in astrocytes and microglia, the inventordeduced that these drugs may have therapeutic potential in correction ofthe metabolic defect and inhibition of the neuroinflammatory diseaseprocess in X-ALD.

The inventor found that PD 98059, an inhibitor of MAP kinase (MEK), thekinase responsible for the activation of MAP kinase, inhibits theLPS-induced activation of NF-kB and the induction of iNOS in astrocytesindicating the possible involvement of the MAP kinase pathway in theinduction of iNOS. MAP kinases exhibit dual-specificity, regulating bothserine (Ser)/threonine (Thr) phosphorylation and Tyr autophosphorylation(Blenis, 1993; Rossomando et al., 1994; Her et al., 1993). In addition,MAP kinases themselves require concurrent Thr and Tyr phosphorylationfor activation, and are, in turn, substrates for MEK (Blenis, 1993;Rossomando et al., 1994; Her et al., 1993). MEK is also a dualspecificity kinase whose activation requires Ser/Thr phosphorylation(Blenis, 1993; Rossomando et al., 1994; Her et al., 1993). The inventordeduced form these observations that cellular regulation of thissignaling pathway may utilize Ser/Thr phosphatases to modulate thephosphorylation state of critical phosphoproteins.

Since phosphoprotein phosphatases (PP) 1 and PP 2A are the two mostabundant Ser/Thr phosphatases in the cell, the study presented inExample 6 was undertaken to investigate the cellular regulation of theinduction of iNOS by PP 1 and PP 2A in rat primary astrocytes andmacrophages. The results clearly demonstrate that calyculin A,microcystin, cantharidin and okadaic acid, inhibitors of PP 1 and PP 2A,stimulate the LPS- and cytokine-mediated expression of iNOS andproduction of NO in astrocytes and C₆ glial cells while the sameinhibitors inhibit the LPS- and cytokine-mediated expression of iNOS andproduction of NO in macrophages and RAW 264.7 cells. Consistent withthis observation, okadaic acid stimulates the iNOS promoter-derivedchloramphenicol acetyl transferase (CAT) activity in LPS-treatedastrocytes but inhibits the iNOS promoter-derived CAT activity inLPS-treated macrophages. This differential regulation of the inductionof iNOS in astrocytes and macrophages by inhibitors of PP 1/2A indicatesthat, although PP 1/2A functions as a physiological inhibitor of theinduction of iNOS in astrocytes, the induction of iNOS in macrophagesrequires the involvement of PP 1/2A. However, in spite of thisdifferential regulation of the induction of iNOS in astrocytes andmacrophages, inhibitors of PP 1/2A stimulate the activation of NF-kB andthe production of TNF-α in both astrocytes and macrophages.

Transient modulation of protein phosphorylation and dephosphorylation isa major mechanism of intracellular signal transduction pathwaystriggered by different cytokines. Therefore, the inventor hypothesizedthat inhibition of protein phosphatase 1 and 2A (PP 1 and 2A) activitieswill influence cytokine induced signal transduction pathways for theinduction of iNOS. The signaling events in cytokine-mediated inductionof iNOS in astrocytes and macrophages are not well understood. Anunderstanding of the cellular mechanisms involved in the induction ofiNOS should identify novel targets for therapeutic intervention inNO-mediated neuroinflammatory diseases. Several lines of evidencepresented in Example 6 support the conclusion that inhibition of PP 1/2Aactivity differentially modulates the LPS- and cytokine-inducedexpression of iNOS and production of NO in rat primary astrocytes andmacrophages. The conclusion is based on the following observations.First, treatment of astrocytes and macrophages with LPS and/or cytokinesinduced the expression of iNOS and production of NO, and inhibitors ofPP 2B (cypermethrin, deltamethrin and fenvalerate) had no effects on theLPS- and cytokine-mediated induction of iNOS and production of NO.Second, compounds (calyculin A, microcystin, okadaic acid andcantharidin) that inhibit PP 1/2A stimulated the LPS- andcytokine-mediated production of NO as well as expression of iNOS proteinand mRNA in astrocytes and C₆ glial cells. However, in contrast, theseinhibitors inhibited the LPS- and cytokine-mediated production of NO andexpression of iNOS in rat resident macrophages and RAW 264.7 cells.Third, the inhibitors of PP 1/2A stimulated iNOS promoter-derivedchloramphenicol acetyl transferase (CAT) activity in LPS-treatedastrocytes but inhibited iNOS promoter-derived CAT activity inLPS-treated macrophages. These results indicate that the signalingevents required for the induction of iNOS in astrocytes differ fromthose required for the induction of iNOS in macrophages.

Cytokines (TNF-α, IL-1β or IFN-γ) and LPS bind to their respectivereceptors and induce iNOS expression via activation of NF-kB (Xie etal., 1994, Kwon et al., 1995). The nuclear expression and biologicalfunction of the NF-kB transcription factor are tightly regulated throughits cytoplasmic retention by the ankyrin-rich inhibitor IkBα (Beg etal., 1992). Activation of NF-kB by various cellular stimuli involves theproteolytic degradation of IkBα and the concomitant nucleartranslocation of the liberated NF-kB heterodimer. Although thebiochemical mechanism underlying the degradation of IkBα remainsunclear, it appears that degradation of IkBα induced by various mitogensand cytokines occurs in association with the transient phosphorylationof IkBα on serines 32 and 36. Further the inventor has found that the 90kDa ribosomal S6 kinase (a downstream candidate of the wellcharacterized Ras-Raf-MEK-MAP kinase pathway), but not p70 S6 kinase orMAP kinase, phosphorylates the N-terminal regulatory domain of IkBα onserine 32. However, in vivo, only phorbol 12-myristate 13-acetateproduced rapid activation of p90 RSK, other potent NF-kB inducersincluding TNF-α and the Tax transactivator of human T-cell lymphotrophicvirus, type I, failed to activate p90 RSK indicating that more than asingle IkBα kinase exists within the cell and that these IkBα kinasesare differentially activated by different NF-kB inducers. Byphosphorylation, IkBα which is still bound to NF-kB has apparentlyturned into a high affinity substrate for an ubiquitin-conjugatingenzyme. Following this phosphorylation-controlled ubiquitination, IkBαis rapidly and completely degraded by the 20 S or 26 S proteosome.

Okadaic acid and other inhibitors of PP 1/2A have also been shown toinduce the activation of NF-kB in monocytes, Jurkat T cells and Helacells (Menon et al., 1993; Suzuke et al., 1994) due to thephosphorylation of IkBα at protein phosphatase 2A-sensitivephosphorylation sites which are different than cytokine-inducedphosphorylation sites (Sun et al., 1995). However, according to Baeuerleand colleagues (Schmidt et al., 1995), okadaic acid-mediated activationof NF-kB in Hela cells requires the induction of oxidative stress.Identification of binding site of NF-kB in the promoter region of iNOSgene and the activation of NF-kB during cytokine-induced iNOS expressionestablishes the role of NF-kB activation in the induction of iNOS (Xieet al., 1994; Kwon et al., 1995). In contrast to the ability of okadaicacid on the activation of NF-kB in other cell types (Menon et al., 1993;Suzuke et al., 1994), okadaic acid by itself was unable to induce theactivation of NF-kB in rat primary astrocytes. However, okadaic acidmarkedly stimulated LPS- or cytokine-mediated activation of NF-kB inastrocytes. Increase in the activation of NF-kB in LPS-stimulatedastrocytes by okadaic acid paralleled the increase in induction of iNOSindicating that stimulation of iNOS expression in LPS-activated ratprimary astrocytes by inhibitors of PP 1/2A is probably mediated viaenhanced activation of NF-kB. However, consistent with the effect ofokadaic acid on the activation of NF-kB in other cell types (Menon etal., 1993; Suzuke et al., 1994), okadaic acid by itself induced theactivation of NF-kB in macrophages but this activation of NF-kB byokadaic acid did not result in the induction of iNOS indicating thatactivation of NF-kB by okadaic acid is not sufficient for the inductionof iNOS in macrophages. Although similar to astrocytes, okadaic acidstimulated the LPS-mediated activation of NF-kB in rat peritonealmacrophages, yet in sharp contrast to the effect of okadaic acid on theinduction of iNOS in astrocytes, the stimulation of NF-kB activation byokadaic acid in LPS-treated macrophages did not parallel with theexpression of iNOS. Instead, consistent with a previous report, okadaicacid and other inhibitors of PP 1/2A markedly inhibited LPS- andcytokine-induced expression of iNOS in macrophages. However, the basisfor this differential regulation of induction of iNOS in astrocytes andmacrophages by inhibitors of PP 1/2A is not understood at the presenttime.

Earlier, the inventor observed that cAMP-dependent protein kinase A(PKA) also differentially modulates the induction of iNOS in astrocytesand macrophages. Inhibition of the activation of NF kB and the inductionof iNOS with the increase in PKA activity, and stimulation of theactivation of NF-kB and the induction of iNOS with the decrease in PP1/2A activities in astrocytes indicate that both PKA (a serine-threonineprotein kinase) and PP 1/2A (serine-threonine phosphoproteinphosphatases) function as inhibitory signals for the induction of iNOSin astrocytes modulating different steps of the signal transductionpathways. In contrast, in macrophages, inhibitors of PKA inhibited theLPS-mediated activation of NF-kB and induction of iNOS, and inhibitorsof PP 1/2A stimulated the LPS-mediated activation of NF-kB but inhibitedthe induction of iNOS indicating that both PKA and PP 1/2A are necessarycomponents of the LPS-mediated signaling pathways for the induction ofiNOS. However, the molecular basis for the differential regulation ofactivation of NF-kB and expression of iNOS gene by inhibitors of PP 1/2Ain rat peritoneal macrophages is not known. In light of the fact thatNF-kB is necessary but not sufficient for the expression of iNOS geneand that many of the signal transduction events are cell type specific,the apparent stimulation of NF-kB and inhibition of iNOS gene expressionby inhibitors of PP 1/2A clearly delineate that apart from theactivation of NF-kB some other signaling pathway(s) sensitive to PP 1/2Ais/are responsible for the expression of iNOS gene in macrophages.

The inventor examined the possible involvement of ROS incytokine-mediated activation of sphingomyelin breakdown and ceramideformation and found that intracellular GSH plays a crucial role in thebreakdown of SM to ceramide, in that low GSH levels are required forceramide generation and high GSH levels inhibit production of ceramide.Inhibition of cytokine-mediated breakdown of SM to ceramide byantioxidants like N-acetyl cysteine (NAC) and pyrrolidinedithiocarbamate (PDTC) and induction of ceramide production by oxidantsor pro-oxidants like hydrogen peroxide, aminotriazole, diamide andL-buthione (S,R)-sulfoximine clearly delineate a novel function of ROSand GSH in regulation of the first step of sphingomyelin signaltransduction pathway. Moreover, decreased levels of GSH and increasedlevels of ceramide correlate with the DNA fragmentation in rat primaryoligodendrocytes as well as in the banked human brains from patientswith neuroinflammatory diseases (e.g. multiple sclerosis andX=adrenoleukodystrophy).

Changes in the cellular redox state toward either prooxidant orantioxidant conditions have profound effects on cellular functions.Several lines of evidence presented herein indicate that the first stepof cytokine-induced sphingomyelin signal transduction pathway (i.e.breakdown of sphingomyelin to ceramide and phosphocholine) is redoxsensitive. First, cytokines like TNF-α and IL-1β decreased intracellularGSH and induced the degradation of sphingomyelin to ceramide in ratprimary astrocytes, oligodendrocytes, microglia and rat C₆ glial cells,and pretreatment of the cells with antioxidants like NAC restored thelevels of GSH and blocked the degradation of sphingomyelin to ceramide.Second, depletion of endogenous glutathione by diamide or buthionesulfoximine alone induces the degradation of sphingomyelin to ceramidewhich is blocked by NAC. Third, the increase in intracellular H₂O₂ bythe addition of exogenous H₂O₂ or by the inhibition of endogenouscatalase by aminotriazole induced the degradation of sphingomyelin toceramide which is also blocked by NAC. Fourth, besides NAC, pyrrolidinedithiocarbamate (PDTC), an amioxidant but not the precursor of GSH(Laight et al., 1997), also inhibited the TNF-α and IL-1β-inducedhydrolysis of sphingomyelin to ceramide.

Several studies support a role for hydrolysis of sphingomyelin as astress-activated signaling mechanism in which ceramide plays a role ingrowth suppression and apoptosis in various cell types including glialand neuronal cells (Brugg et al., 1996; Wiesner and Dawson, 1996).Ceramide activates the proteases of the interleukin converting enzyme(ICE) family, (especially prICE/YAMA/CPP32), the protease responsiblefor cleavage of poly-ADP-ribose polymerase (PARP) (Martin et al., 1995)and that the activation of prICE by ceramide and induction of apoptosisare inhibited by overexpression of Bcl-2 (Zhang et al., 1996). Additionof exogenous ceramides or sphingomyelinase to cells induces stressactivated protein kinase (SAPK)-dependent transcriptional activitythrough the activation of c jun (Latinis and Koretzky, 1996) and adominant negative mutant of SEK1, the protein kinase responsible forphosphorylation and activation of SAPK, interferes with ceramide-inducedapoptosis (Verheij et al, 1996). These observations also indicate thatboth Bcl-2 and SAPK function downstream of ceramide in the apoptoticpathway.

The inventor has found that DNA fragmentation and increase in ceramideand decrease in GSH in primary oligodendrocytes and banked human brainswith X-ALD and MS clearly indicate that intracellular redox (level ofGSH) is an important regulator of apoptosis via controlling thegeneration of ceramide. This conclusion is based on followingobservations. First, treatment of oligodendrocytes with TNF-α decreasedintracellular level of GSH, increased degradation of SM to ceramide andinduced DNA fragmentation, however, pretreatment of oligodendrocyteswith NAC blocked the TNF-α-mediated decrease in GSH level, increase inceramide level and increase in DNA fragmentation. Second, treatment ofoligodendrocytes only with diamide, a thiol-depleting agent, decreasedintracellular level of GSH, increased level of ceramide and induced DNAfragmentation which are prevented by pretreatment of NAC, athiol-replenishing agent. Third, the inventor found increasedfragmentation of DNA in brains from patients with X-ALD and MS where thelevels of GSH and ceramide were lower and higher respectively comparedto those found in control human brains. These observations clearlyindicate that maintenance of the thiol/oxidant balance is crucial forprotection against cytokine-mediated ceramide production and therebyagainst ceramide-induced cytotoxicity.

Recent observation demonstrated that ceramide potentiates thecytokine-mediated induction of inducible nitric oxide synthase (iNOS) inastrocytes and C₆ glial cells. Although ceramide by itself did notinduce the expression of iNOS and production of NO, it markedlystimulated the cytokine-induced expression of iNOS and production of NOindicating that sphingomyelin-derived ceramide generation may be animportant factor in cytokine-mediated cytotoxicity in neurons andoligodendrocytes in neuroinflammatory diseases. The N-acetyl cysteine(NAC), which has been used to block the cytokine-induced ceramideproduction in this study and to inhibit cytokine-mediated induction ofiNOS is a nontoxic pharmaceutical drug that enters the cell readily andserves both as a scavenger of ROS and a precursor of GSH, the majorintracellular thiol (Smilkstein et al., 1988). Therefore, the use ofreductants such as NAC or other thiol compounds, may be beneficial inrestoring cellular redox and in inhibition of cytokine-mediatedinduction of iNOS and breakdown of sphingomyelin thus reducingNO-mediated cytotoxicity as well as ceramide-mediated apoptosis inneuroinflammatory diseases.

Inhibitors, Enhancers and Screening Assays

In still further embodiments, the present invention provides methods foridentifying new iNOS and/or proinflammatory cytokine inhibitorycompounds, which may be termed as “candidate substances.” It iscontemplated that such screening techniques will prove useful in thegeneral identification of any compound that will serve the purpose ofinhibiting iNOS and/or proinflammatory cytokines, and in preferredembodiments, will provide candidate therapeutic compounds. The presentinvention also provides methods for identifying new iNOS and/orproinflammatory cytokine stimulatory or enhancing compounds.

It is further contemplated that useful compounds in this regard will inno way be limited to proteinaceous or peptidyl compounds. In fact, itmay prove to be the case that the most useful pharmacological compoundsfor identification through application of the screening assays will benon-peptidyl in nature and, e.g., which will serve to inhibit or enhanceiNOS and/or proinflammatory cytokine activity or transcription through atight binding or other chemical interaction. Candidate substances may beobtained from libraries of synthetic chemicals, or from natural samples,such as rain forest and marine samples.

In preferred embodiments, a candidate induction suppressor and/orinhibitor of inducible nitric oxide synthase and/or proinflammatorycytokines is selected from agents that have certain traits or modes ofaction common to those of the suppressors and/or inhibitors identifiedherein. Preferred candidate substances would either inhibit theRas/Raf/MAP kinase pathway, inhibit and/or suppress the induction and/oractivation of NF-kB, inhibit mevalonate synthesis, be an enhancer ofprotein kinase A, and/or inhibit the farnasylation of proteins,including but not limited to Ras. In certain embodiments the inhibitorof mevalonate synthesis may be an inhibitor of HMG-CoA reductase orsuppressor of its induction. In certain aspects the inhibitor of HMG-CoAreductase is a stimulator of AMP-activated protein kinase. In certainother embodiments the inhibitor of inducible nitric oxide synthaseand/or proinflammatory cytokines may be an inhibitor of mevalonatepyrophosphate decarboxylase or suppressor of its induction. In otherembodiments the candidate substance is an antioxidant. In otherembodiments the candidate substance is an enhancer of intracellularcAMP. The enhancer of intracellular cAMP may be an inhibitor of cAMPphosphodiesterase and/or suppressor of its induction. In otherembodiments the candidate substance is a farnesyl protein transferaseinhibitor, and/or induction suppressor.

In other preferred embodiments, a candidate induction suppressor and/orinhibitor of inducible nitric oxide synthase and/or proinflammatorycytokines is selected from agents that have certain traits or modes ofaction common to those of the stimulators and/or enhancers identifiedherein. For example, a preferred candidate stimulators or enhancerswould include a PKA inhibitor.

In other embodiments, the present invention provides methods foridentifying new iNOS and/or proinflammatory cytokine inhibitory orstimulatory compounds. To determine whether a candidate substance hasinhibitory, suppressor, stimulator, or enhancer activity for iNOS,and/or proinflammatory cytokines, assays may be employed to detect ormeasure the change in the message, content, and/or activity of iNOS,proinflammatory cytokines such as TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ, proteins involved in second messenger pathways, or transcriptionfactors such as NF-kβ.

Nucleic Acid Detection

Assays for the detection of iNOS, NF-kβ, and/or proinflammatorycytokines such as TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ includedetection of changes in the amount of nucleic acid message in a cellupon contact with a candidate inhibitor, suppressing agent, enhancer,and/or stimulator. Such assays are described in the specific examples.Additionally, gene sequences for known iNOS, NF-kβ, and/orproinflammatory cytokines in a database such as found in the NationalCenter for Biotechnology Information (internet web site:http://www.ncbi.nlm.nih.gov) may be used as probes or primers in nucleicacid hybridization embodiments of such assays.

1. Hybridization

The use of a hybridization probe of between 17 and 100 nucleotides inlength, or in some aspect of the invention even up to 1-2 Kb or more inlength, allows the formation of a duplex molecule that is both stableand selective. Molecules having complementary sequences over stretchesgreater than 20 bases in length are generally preferred, in order toincrease stability and selectivity of the hybrid, and thereby improvethe quality and degree of particular hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules having stretches of 20to 30 nucleotides, or even longer where desired. Such fragments may bereadily prepared by, for example, directly synthesizing the fragment bychemical means or by introducing selected sequences into recombinantvectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of genes or RNAs or to provide primers for amplification ofDNA or RNA from tissues. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.10 M NaCl at temperatures ofabout 50° C. to about 70° C. Such high stringency conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for isolating specific genesor detecting specific mRNA transcripts. It is generally appreciated thatconditions can be rendered more stringent by the addition of increasingamounts of formamide.

For certain applications, for example, substitution of nucleotides bysite-directed mutagenesis, it is appreciated that lower stringencyconditions are required. Under these conditions, hybridization may occureven though the sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of being detected. In preferred embodiments, one may desireto employ a fluorescent label or an enzyme tag such as urease, alkalinephosphatase or peroxidase, instead of radioactive or otherenvironmentally undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a detection means visible to the human eye orspectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization, as inPCR™, for detection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The selected conditions will depend on the particularcircumstances based on the particular criteria required (depending, forexample, on the G+C content, type of target nucleic acid, source ofnucleic acid, size of hybridization probe, etc.). Following washing ofthe hybridized surface to remove non-specifically bound probe molecules,hybridization is detected, or even quantified, by means of the label.

2. Amplification and PCR™

Nucleic acid used as a template for amplification is isolated from cellscontained in the biological sample, according to standard methodologies(Sambrook et al., 1989). The nucleic acid may be genomic DNA orfractionated or whole cell RNA. Where RNA is used, it may be desired toconvert the RNA to a complementary DNA. In one embodiment, the RNA iswhole cell RNA and is used directly as the template for amplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ genes are contacted with the isolated nucleic acid underconditions that permit selective hybridization. The term “primer”, asdefined herein, is meant to encompass any nucleic acid that is capableof priming the synthesis of a nascent nucleic acid in atemplate-dependent process. Typically, primers are oligonucleotides fromten to twenty or thirty base pairs in length, but longer sequences canbe employed. Primers may be provided in double-stranded orsingle-stranded form, although the single-stranded form is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with oneor more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In certain applications,the detection may be performed by visual means. Alternatively, thedetection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (Affymax technology).

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, each incorporated herein by reference inentirety.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin WO 90/07641, filed Dec. 21, 1990, incorporated herein by reference.Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880,incorporated herein by reference, may also be used as still anotheramplification method in the present invention. In this method, areplicative sequence of RNA that has a region complementary to that of atarget is added to a sample in the presence of an RNA polymerase. Thepolymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Gingeras et al., PCT Application WO88/10315, incorporated herein by reference). In NASBA, the nucleic acidscan be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer which has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double stranded by addition of secondtarget specific primer, followed by polymerization. The double-strandedDNA molecules are then multiply transcribed by an RNA polymerase such asT7 or SP6. In an isothermal cyclic reaction, the RNA's are reversetranscribed into single stranded DNA, which is then converted to doublestranded DNA, and then transcribed once again with an RNA polymerasesuch as T7 or SP6. The resulting products, whether truncated orcomplete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H(RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large. “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR™” (Frohman, 1990, incorporated herein by reference).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989).

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridizationwith a labeled probe. The techniques involved in Southern blotting arewell known to those of skill in the art and can be found in manystandard books on molecular protocols. See Sambrook et al., 1989.Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and non-covalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

All the essential materials and reagents required for changes in iNOSand/or proinflammatory cytokines in a biological sample may be assembledtogether in a kit. This generally will comprise preselected primers forspecific markers. Also included may be enzymes suitable for amplifyingnucleic acids including various polymerases (RT, Taq, etc.),deoxynucleotides and buffers to provide the necessary reaction mixturefor amplification. Such kits generally will comprise, in suitable means,distinct containers for each individual reagent and enzyme as well asfor each marker primer pair.

In another embodiment, such kits will comprise hybridization probesspecific for iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ.Such kits generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachmarker hybridization probe.

Immunodetection Methods

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifying orotherwise generally detecting biological components such as iNOS, NF-kβ,TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ components. The antibodiesspecific for iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γmay be prepared in accordance with the present invention may be employedto detect wild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6,IL-8 and/or IFN-γ proteins, polypeptides or peptides. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Nakamura et al. (1987),incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing an iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8and/or IFN-γ protein, polypeptide or peptide, and contacting the samplewith a first anti-iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ antibody in accordance with the present invention, as the case maybe, under conditions effective to allow the formation ofimmunocomplexes.

These methods include methods for purifying wild-type or mutant iNOS,NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ proteins,polypeptides or peptides as may be employed in purifying wild-type ormutant iNOS, NF-k TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ proteins,polypeptides or peptides from patients' samples or for purifyingrecombinantly expressed wild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β,IL-2, IL-6, IL-8 and/or IFN-γ proteins, polypeptides or peptides. Inthese instances, the antibody removes the antigenic wild-type or mutantiNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ protein,polypeptide or peptide component from a sample. The antibody willpreferably be linked to a solid support, such as in the form of a columnmatrix, and the sample suspected of containing the wild-type or mutantiNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ antigeniccomponent will be applied to the immobilized antibody. The unwantedcomponents will be washed from the column, leaving the antigenimmunocomplexed to the immobilized antibody, which wild-type or mutantiNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ antigen is thencollected by removing the wild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β,IL-2, IL-6, IL-8 and/or IFN-γ from the column.

The immunobinding methods also include methods for detecting orquantifying the amount of a wild-type or mutant iNOS, NF-kβ, TNF-α,IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ protein reactive component in asample, which methods require the detection or quantification of anyimmune complexes formed during the binding process. Here, one wouldobtain a sample suspected of containing a wild-type or mutant iNOS,NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ protein, polypeptideor peptide, and contact the sample with an antibody against wild-type ormutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ, andthen detect or quantify the amount of immune complexes formed under thespecific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing a wild-type or mutant iNOS,NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ protein-specificantigen, such as a lymphocytes, macrophages, endothelial cells,astrocytes, microglia, oligodendrocytes and/or neuron tissue section orspecimen, a homogenized lymphocytes, macrophages, endothelial cells,astrocytes, microglia, oligodendrocytes and/or neuron tissue extract, oreven any biological fluid that comes into contact with diseasedlymphocytes, macrophages, endothelial cells, astrocytes, microglia,oligodendrocytes and neurons tissue, including blood and serum, althoughtissue samples and extracts are preferred.

Contacting the chosen biological sample with the antibody underconditions effective and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time lone enough for theantibodies to form immune complexes with, i.e., to bind to, any iNOS,NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ antigens present.After this time, the sample-antibody composition, such as a tissuesection, ELISA plate, dot blot or western blot, will generally be washedto remove any non-specifically bound antibody species, allowing onlythose antibodies specifically bound within the primary immune complexesto be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological or enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody or abiotin/avidin ligand binding arrangement, as is known in the art.

The iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ antibodyemployed in the detection may itself be linked to a detectable label,wherein one would then simply detect this label, thereby allowing theamount of the primary immune complexes in the composition to bedetermined. Alternatively, the first antibody that becomes bound withinthe primary immune complexes may be detected by means of a secondbinding ligand that has binding affinity for the antibody. In thesecases, the second binding ligand may be linked to a detectable label.The second binding ligand is itself often an antibody, which may thus betermed a “secondary” antibody. The primary immune complexes arecontacted with the labeled, secondary binding ligand, or antibody, underconditions effective and for a period of time sufficient to allow theformation of secondary immune complexes. The secondary immune complexesare then generally washed to remove any non-specifically bound labeledsecondary antibodies or ligands, and the remaining label in thesecondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under conditionseffective and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

In the detection of an alteration in the levels of iNOS, NF-kβ, TNF-α,IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ gene message, translation product,and/or activity in or from a biological sample, such as a cell, tissue,or organism, a comparison is made between a biological sample uponcontact with a candidate suppressor, inhibitor, stimulator, and/orenhancer, to that of a similar or like biological sample that has notcontacted with a candidate suppressor, inhibitor, stimulator, and/orenhancer. Reduced levels of iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8and/or IFN-γ gene message, translation product, and/or activity of acell or patient is indictictive of the candidate substance being ainhibitor or suppressor. An enhancer or stimulator would be identifiedby increased levels of iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8and/or IFN-γ gene message, translation product, and/or activity.Preferably, the biological sample has been contacted with a knowninducer or enhancer, such as LPS and/or proinflammatory cytokines, or asuppressor or inhibitor, either before, during, and/or after contactwith the candidate substance, to help measure the candidate substance'seffect on the activity of the known inducer, suppressor, inhibitor, orenhancer. Those of skill in the art are very familiar withdifferentiating between significant differences in types or amounts ofiNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ, whichrepresent a positive identification, and low level or background changesof iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ. Indeed,background expression levels are often used to form a “cut-off” abovewhich increased detection will be scored as significant or positive. Inthis case, “background” levels may be the levels of iNOS, NF-kβ, TNF-α,IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ seen after stimulation of a cell orpatient with endotoxin and/or a cytokine, preferably a proinflammatorycytokine.

1. ELISAs

As detailed above, immunoassays, in their most simple and direct sense,are binding assays. Certain preferred immunoassays are the various typesof enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays(RIA) known in the art. Immunohistochemical detection using tissuesections is also particularly useful. However, it will be readilyappreciated that detection is not limited to such techniques, andwestern blotting, dot blotting, FACS analyses, and the like may also beused.

In one exemplary ELISA, the anti-iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6,IL-8 and/or IFN-γ antibodies are immobilized onto a selected surfaceexhibiting protein affinity, such as a well in, a polystyrene microtiterplate. Then, a test composition suspected of containing the wild-type ormutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ proteinantigen, such as a clinical sample, is added to the wells. After bindingand washing to remove non-specifically bound immune complexes, the boundwild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ protein antigen may be detected. Detection is generally achievedby the addition of another anti-iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6,IL-8 and/or IFN-γ antibody that is linked to a detectable label. Thistype of ELISA is a simple “sandwich ELISA”. Detection may also beachieved by the addition of a second anti-iNOS, NF-kβ, TNF-α, IL-1β,IL-2, IL-6, IL-8 and/or IFN-γ antibody, followed by the addition of athird antibody that has binding affinity for the second antibody, withthe third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing thewild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ antigen are immobilized onto the well surface and then contactedwith the anti-iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γantibodies of the invention. After binding and washing to removenon-specifically bound immune complexes, the bound anti-iNOS, NF-kβ,TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ antibodies are detected.Where the initial anti-iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8and/or IFN-γ antibodies are linked to a detectable label, the immunecomplexes may be detected directly. Again, the immune complexes may bedetected using a second antibody that has binding affinity for the firstanti-iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ antibody,with the second antibody being linked to a detectable label.

Another ELISA in which the wild-type or mutant iNOS, NF-kβ, TNF-α,IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ proteins, polypeptides or peptidesare immobilized, involves the use of antibody competition in thedetection. In this ELISA, labeled antibodies against wild-type or mutantiNOS, NF-kβ, TNF-α, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/or IFN-γ proteinare added to the wells, allowed to bind, and detected by means of theirlabel. The amount of wild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β,IL-2, IL-6, IL-8 and/or IFN-γ protein antigen in an unknown sample isthen determined by mixing the sample with the labeled antibodies againstwild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ before or during incubation with coated wells. The presence ofwild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8 and/orIFN-γ protein in the sample acts to reduce the amount of antibodyagainst wild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8and/or IFN-γ protein available for binding to the well and thus reducesthe ultimate signal. This is also appropriate for detecting antibodiesagainst wild-type or mutant iNOS, NF-kβ, TNF-α, IL-1β, IL-2, IL-6, IL-8and/or IFN-γ protein in an unknown sample, where the unlabeledantibodies bind to the antigen-coated wells and also reduces the amountof antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein and solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, or a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact andincubate the first or second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., by,incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer.

2. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). The method ofpreparing tissue blocks from these particulate specimens has beensuccessfully used in previous IHC studies of various prognostic factors,and is well known to those of skill in the art (Brown et al., 1990;Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule andremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and embedding the block in paraffin; and cutting up to 50serial permanent sections.

Second Generation Inhibitors or Enhancers

In addition to the inhibitory compounds initially identified, theinventor also contemplates that other sterically similar compounds maybe formulated to mimic the key portions of the structure of theinhibitors and/or enhancers. Such compounds, which may includepeptidomimetics of peptide inhibitors and/or enhancer, may be used inthe same manner as the initial inhibitors and/or enhancers.

Certain mimetics that mimic elements of protein secondary structure aredesigned using the rationale that the peptide backbone of proteinsexists chiefly to orientate amino acid side chains in such a way as tofacilitate molecular interactions. A peptide mimetic is thus designed topermit molecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms, as is well known to the skilled artisan.Once the component amino acids of the turn are determined, mimetics canbe constructed to achieve a similar spatial orientation of the essentialelements of the amino acid side chains.

The generation of further structural equivalents or mimetics may beachieved by the techniques of modeling and chemical design known tothose of skill in the art. The art of computer-based chemical modelingis now well known. Using such methods, a chemical that specificallyinhibits iNOS and/or proinflammatory cytokines can be designed, and thensynthesized, following the initial identification of a compound thatinhibits iNOS and/or proinflammatory cytokines activity and/orinduction, but that is not specific or sufficiently specific to inhibitiNOS activity in individuals suffering from demylenating diseases orneural trauma. Also using such methods, a chemical that specificallyenhances iNOS and/or proinflammatory cytokines can be designed, and thensynthesized, following the initial identification of a compound thatenhances iNOS and/or proinflammatory cytokines activity and/orinduction. It will be understood that all such sterically similarconstructs and second generation molecules fall within the scope of thepresent invention.

Optimization in Therapy

A compound identified as having the ability to inhibit or enhance theinduction of iNOS and/or cytokines can be assayed its optimumtherapeutic dosage alone or in combination with another anti-iNOS,anti-cytokine or anti-inflammatory agent. Such assays are well known tothose of skill in the art, and include tissue culture or animal modelsfor various disorders that are treatable with such agents.

Examples of such assays include those described herein and in U.S. Pat.No. 5,696,109, the disclosure of which is incorporated herein byreference in its entirety. For instance, an assay to determine thetherapeutic potential of molecules in brain ischemia (stroke) evaluatesan agent's ability to prevent irreversible damage induced by an anoxicepisode in brain slices maintained under physiological conditions. Ananimal model of Parkinson's disease involving iatrogenic hydroxylradical generation by the neurotoxin MPTP (Chiueh et al., 1992,incorporated herein by reference) may be used to evaluate the protectiveeffects of iNOS or pro-inflammatory cytokine induction inhibitors. Theneurotoxin, MPTP, has been shown to lead to the degeneration ofdopaminergic neurons in the brain, thus providing a good model ofexperimentally induced Parkinson's disease (e.g. iatrogenic toxicity).An animal model of ischemia and reperfision damage is described usingisolated iron-overloaded rat hearts to measure the protective ortherapeutic benefits of an agent. Briefly, rats receive an intramuscularinjection of an iron-dextran solution to achieve a significant ironoverload in cardiac tissue. Heart are then isolated and then subjectedto total global normothermic ischemia, followed by reperfusion with theperfusion medium used initially. During this reperfusion, heart rate,and diastolic and systolic pressures were monitored. Cardiac tissuesamples undergo the electron microscopy evaluation to measure damage tomitochondria such as swelling and membrane rupture, and cell necrosis.Comparison of measured cardiac function and cellular structural damagewith or without the agent or iron-overloading afterischemia/reoxygenation is used to determine the therapeuticeffectiveness of the agent. Another assay measures acute lung injury(ALI) in sepsis and endotoxemia. LPS/endotoxin-induced ALI in pigs maybe used as a model to measure the effectiveness of an agent for thetreatment of sepsis-induced ALI in humans. After infusion ofLPS/endotoxin, changes in lung wet-to-dry weight ratio, lung lipidperoxidation, pulmonary arterial hypertension, arterial hypoxemia anddecreased dynamic pulmonary compliance is measured to determine theeffectiveness of an agent in preventing LPS/endotoxin induced damage.

One of skill in the art will recognize that there are other assays andmodels for disease states available, including testing in humans. Theseassays may be used to measure the effectiveness of iNOS and/orpro-inflammatory cytokine induction suppressor and/or inhibitor agentfor a particular disease or condition, determine the best agent orcombination of agents to be used, and determine the dosages foradministration, with routine experimentation.

Combination Therapy

The suppressor agents of the invention may also be used in combinationwith other therapeutic agents, for example, anti-inflammatory agents,particularly non-steroidal anti-inflammatory drugs (NSAIDs), vasodilatorprostaglandins including prostacyclin and prostaglandin E sub 1, cancerchemotherapeutic agents including cisplatin, NO donors or NO inhalationtherapy, or PAF-receptor antagonists.

Pharmaceutical Compositions

A further aspect of the invention are compositions comprising a firstiNOS and/or proinflammatory cytokine inhibitor and/or inductionsuppressor in a pharmaceutically-acceptable excipient. In a preferredembodiment, the iNOS and/or proinflammatory cytokine inhibitor and/orinduction suppressor is selected from the group consisting oflovastatin, mevastatin, FPT inhibitor II, forskolin, rolipram,phenylacetate (NaPA), N-acetyl cysteine (NAC), PDTC, 4-phenylbutyrate(4PBA), 5-aminoimmidazole-4-carboxamide ribonucleoside (AICAR),theophylline, papaverine, cAMP, 8-bromo-cAMP, (S)-cAMP, and salts,analogs, or derivatives thereof.

A further aspect of the invention is a composition which comprises atleast two iNOS and/or proinflammatory cytokine inhibitor and/orinduction suppressor in a pharmaceutically-acceptable excipient. In apreferred embodiment, at least one of the iNOS and/or proinflammatorycytokine inhibitors or suppressors is selected from the group consistingof lovastatin, mevastatin, FPT inhibitor II, forskolin, rolipram,phenylacetate (NaPA), N-acetyl cysteine (NAC), PDTC, 4-phenylbutyrate(4PBA), 5-aminoimmidazole-4-carboxamide ribonucleoside (AICAR),theophylline, papaverine, cAMP, 8-bromo-cAMP, (S)-cAMP, and salts,analogs, or derivatives thereof.

A further aspect of the invention are compositions comprising a firstiNOS and/or proinflammatory cytokine stimulator, enhancer, or inducer ina pharmaceutically-acceptable excipient. A further aspect of theinvention is a composition which comprises at least two iNOS and/orproinflammatory cytokine stimulator, enhancer, or inducer in apharmaceutically-acceptable excipient. In another embodiment, theenhancer, stimulator or inducer of iNOS or proinflammatory cytokines isH-89, myristoylated PKI, (R)-cAMP, forskolin, 8-bromo-cAMP, rolipram andsalts, analogs, or derivatives thereof. Inducers, stimulators orenhancers of iNOS and/or proinflammatory cytokines may be tissuespecific, and such tissues include microglia cells.

In certain embodiments, the suppressors, inhibitors, stimulators,enhancers and/or inducers of iNOS and/or proinflammatory cytokines maybe administered of from about 0.001 mg per kg body weight per day(mg/kg/day) to about 20 mg/kg/day. Of course it is understood that offrom about 0.001 mg/kg/day to about 20 mg/kg/day includes doses of fromabout 0.001, about 0.002, about 0.003, about 0.004, about 0.005, about0.006, about 0.007, about 0.008, about 0.009, about 0.01, about 0.011,about 0.012, about 0.013, about 0.014, about 0.015, about 0.016, about0.017, about 0.018, about 0.019, about 0.02, about 0.021, about 0.022,about 0.023, about 0.024, about 0.025, about 0.026, about 0.027, about0.028, about 0.029, about 0.03, about 0.032, about 0.034, about 0.036,about 0.038, about 0.04, about 0.042, about 0.044, about 0.046, about0.048, about 0.05, about 0.055, about 0.06, about 0.065, about 0.07,about 0.075, about 0.08, about 0.085, about 0.09, about 0.095, about0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about0.46, about 0.47, about 0.48, about 0.49, about 0.50, about 0.51, about0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about0.58, about 0.59, about 0.60, about 0.65, about 0.70, about 0.75, about0.80, about 0.85, about 0.90, about 0.95, about 1.00, about 1.05, about1.10, about 1.15, about 1.20, about 1.25, about 1.30, about 1.35, about1.40, about 1.45, about 1.50; about 1.55, about 1.60, about 1.65, about1.70, about 1.75, about 1.80, about 1.85, about 1.90, about 1.95, about2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about3.20, about 3.30, about 3.40, about 3.50, about 3-60, about 3.70, about3.80, about 3.90, about 4.00, about 4.10, about 4.20, about 4.30, about4.40, about 4.50, about 4.60, about 4.70, about 4.80, about 4.90, about5.00, about 5.25, about 5.5, about 5.75, about 6.00, about 6.25, about6.5, about 6.75, about 7.0, about 7.25, about 7.5, about 7.75, about8.00, about 8.25, about 8.5, about 8.75, about 9.0, about 9.25, about9.5, about 9.75, about 10.00, about 10.5, about 11.0 about 11.5, about12.00, about 12.5, about 13, about 13.5, about 14.00, about 14.5, about15, about 15.5, about 16, about 16.5, about 17.0, about 17.5, about18.00, about 18.5, about 19.0, about 19.5, and about 20.00 mg/kg/day.One may select any dosages described herein as a range of dosageadministration, such as a range of about 1.45 mg/kg/day to about 11mg/kg/day, or about 0.24 mg/kg/day to about 14 mg/kg/day, etc., as wellas any values within such ranges that are not specifically recited.

One of skill in the art will recognize that the toxicity for differentsuppressors, inhibitors, enhancer, stimulator and/or inducers of iNOSand/or proinflammatory cytokines either alone, in combination with eachother, or in combination with other pharmaceuticals may limit themaximum dose administered to a patient. Dosage optimization for maximumbenefits with minimal toxicity in a patient may be optimized by those ofskill in the art without undue experimentation using any method todetermine optimum dosage in a patient as is known to those of the art,or using the methods described herein. Additionally, the suppressors,inhibitors, enhancer, stimulator and/or inducers of the presentinvention may be obtained from commercial vendors and administered inany of the methods or dosages described in exemplary texts, such as“Remington's Pharmaceutical Sciences” 8th and 15th Editions; the“Physicians'Desk Reference”, 1998 Edition, the Merck Index, 11thEdition, each incorporated herein in their entirety).

In certain preferred embodiments, lovastatin or mevastatin is takenorally with food once daily at about 0.01 mg per kg body weight per day(mg/kg/day) to about 0.24 mg/kg/day. Of course it is understood thatabout 0.01 mg/kg/day to about 0.24 mg/kg/day includes doses of about0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about0.19, about 0.20, about 0.21, about 0.22, about 0.23, to about 0.24mg/kg or so per day. In a preferred embodiment, lovastatin or mevastatinis taken orally with food once daily at about 0.25 mg per kg body weightper day (mg/kg/day) to about 0.55 mg/kg/day. Of course it is understoodthat about 0.25 mg/kg/day to about 0.55 mg/kg/day includes doses ofabout 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30,about 0.31, about 0.32, about 0.33, about 0.33, about 0.34, about 0.35,about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41,about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47,about 0.48, about 0.49, about 0.50, about 0.51, about 0.52, about 0.53,about 0.54, to about 0.55 mg/kg or so per day. When two or more iNOSand/or proinflammatory cytokine inhibitors and/or induction suppressorsare administered, the combined dose of two or more iNOS and/orproinflammatory cytokine inhibitors and/or induction suppressors ispreferably about 0.25 mg/kg/day to about 0.55 mg/kg/day. It is alsospecifically contemplated by the inventor that a patient may be treatedwith about 0.55 mg/kg/day to about 5 mg per day or more of lovastatinand/or mevastatin, including about 0.60, about 0.65, about 0.70, about0.75, about 0.80, about 0.85, about 0.90, about 0.95, about 1.00, about1.05, about 1.10, about 1.15, about 1.20, about 1.25, about 1.30, about1.35, about 1.40, about 1.45, about 1.50, about 1.55, about 1.60, about1.65, about 1.70, about 1.75, about 1.80, about 1.85, about 1.90, about1.95, about 2.00, about 2.05, about 2.10, about 2.15, about 2.20, about2.25, about 2.30, about 2.35, about 2.40, about 2.45, about 2.50, about2.55, about 2.60, about 2.65, about 2.70, about 2.75, about 2.80, about2.85, about 2.90, about 2.95, about 3.00, about 3.10, about 3.20, about3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about3.90, about 4.00, about 4.10, about 4.20, about 4.30, about 4.40, about4.50, about 4.60, about 4.70, about 4.80, about 4.90, to about 5.00mg/kg or more per day. Compositions for such treatment are described in,for example U.S. Pat. Nos. 3,983,140, and 4,231,938, the disclosures ofwhich are incorporated herein by reference in their entirety.

In yet another preferred embodiment of the invention nitric oxideinduced cytotoxicity may be prevented or reduced in a patient bytreatment with from about 0.01 mg/kg/day to about 2.0 mg/kg/day ofrolipram, including about 0.01, about 0.02, about 0.04, about 0.06,about 0.08, about 0.10, about 0.12, about 0.14, about 0.16, about 0.18,about 0.20, about 0.22, about 0.24, about 0.26, about 0.28, about 0.30,about 0.32, about 0.34, about 0.36, about 0.38, about 0.40, about 0.42,about 0.44, about 0.46, about 0.48, about 0.50, about 0.52, about 0.54,about 0.56, about 0.58, about 0.60, about 0.62, about 0.64, about 0.66,about 0.68, about 0.70, about 0.72, about 0.74, about 0.76, about 0.78,about 0.80, about 0.82, about 0.84, about 0.86, about 0.88, about 0.90,about 0.92, about 0.94, about 0.96, about 0.98, about 1.00, about 1.01,about 1.02, about 1.04, about 1.06, about 1.08, about 1.10, about 1.12,about 1.14, about 1.16, about 1.18, about 1.20, about 1.22, about 1.24,about 1.26, about 1.28, about 1.30, about 1.32, about 1.34, about 1.36,about 1.38, about 1.40, about 1.42, about 1.44, about 1.46, about 1.48,about 1.50, about 1.52, about 1.54, about 1.56, about 1.58, about 1.60,about 1.62, about 1.64, about 1.66, about 1.68, about 1.70, about 1.72,about 1.74, about 1.76, about 1.78, about 1.80, about 1.82, about 1.84,about 1.86, about 1.88, about 1.90, about 1.92, about 1.94, about 1.96,about 1.98, to about 1.00 or more mg/kg/day. Preferably 0.1 mg/kg/day toabout 0.7 mg/kg/day is used and most preferably about 0.5 mg/kg/day.Compositions for such treatment have been described in, for example,U.S. Pat. No. 5,672,622, specifically incorporated herein by referencein its entirety.

In yet another preferred embodiment of the invention, nitric oxideinduced cytotoxicity may be prevented or reduced in a patient bytreatment with about 0.01 mg/kg/day to about 1.0 mg/kg/day of forskolin,including about 0.01, about 0.02, about 0.04, about 0.06, about 0.08,about 0.10, about 0.12, about 0.14, about 0.16, about 0.18, about 0.20,about 0.22, about 0.24, about 0.26, about 0.28, about 0.30, about 0.32,about 0.34, about 0.36, about 0.38, about 0.40, about 0.42, about 0.44,about 0.46, about 0.48, about 0.50, about 0.52, about 0.54, about 0.56,about 0.58, about 0.60, about 0.62, about 0.64, about 0.66, about 0.68,about 0.70, about 0.72, about 0.74, about 0.76, about 0.78, about 0.80,about 0.82, about 0.84, about 0.86, about 0.88, about 0.90, about 0.92,about 0.94, about 0.96, about 0.98, and about 1.0 or more mg/kg/day.Compositions for such treatment have been described in, for example,U.S. Pat. No. 5,371,104, incorporated herein by reference in itsentirety.

In still yet another preferred embodiment of the invention, nitric oxideinduced cytotoxicity may be prevented or reduced in a patient bytreatment with about 0.1 mg/kg/day to about 20 mg/kg/day of a farnesylprotein transferase inhibitor, for example, FPT II. Specificallycontemplated is any dose within this range, including about 0.1, about0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5,about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5; about 10.0,about 10.5, about 71.0, about 11.5, about 12.0, about 12.5, about 13.0,about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0,about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0,about 19.5 and 20.0 or more mg/kg/day. Preferably about 0-5 mg/kg/day to10 mg/kg/day is used. Compositions for such treatment are described in,for example, U.S. Pat. No. 5,420,157, specifically incorporated hereinby reference in its entirety.

In still yet another preferred embodiment of the invention, nitric oxideinduced cytotoxicity may be prevented or reduced in a patient bytreatment with up to about 50 mg/kg/day of N-acetyl cysteine. Of course,it will be understood that up to about 50 mg/kg/day includes all dosagesdescribed above generically for the iNOS and/or proinflammatory cytokineinhibitors and/or induction suppressors of the present invention, anddosages of from about 20 mg/kg/day to about 50 mg/kg/day, including offrom about 20, about 20.5, about 21, about 21.5, about 22.0, about 22.5,about 23, about 23.5, about 24, about 24.5, about 25, about 25.5, about26, about 26.5, about 27, about 27.5, about 28, about 28.5, about 29,about 29.5, about 30, about 30.5, about 31.5, about 32, about 32.5,about 33, about 33.5, about 34, about 34.5, about 35, about 35.5, about36, about 36.5, about 37, about 37.5, about 38, about 38.5, about 39,about 39.5, about 40, about 40.5, about 41, about 41.5, about 42, about42.5, about 43, about 43.5, about 44, about 44.5, about 45, about 45.5,about 46, about 46.5, about 47, about 47.5, about 48, about 48.5, about49, about 49.5, and about 50.0 or more mg/kg/day. Specific compositionsfor such treatment are disclosed in, for example, U.S. Pat. No.5,080,960, incorporated herein by reference in its entirety.

TABLE 1 Contemplated Ranges for Dose Administration in the Methods ofthe Invention Lovastatin Range About 0.001 mg/kg/day to about 20.00mg/kg/day Preferred Range About 0.01 to about 5.00 mg/kg/day MorePreferred About 0.25 to about 0.55 mg/kg/day Range Mevastatin RangeAbout 0.001 mg/kg/day to about 20.00 mg/kg/day Preferred Range About0.01 to about 5.00 mg/kg/day More Preferred About 0.25 to about 0.55mg/kg/day Range Phenyl Acetic Acid Range About 0.001 mg/kg/day to about20.00 mg/kg/day N-acetyl Cysteine Range About 0.001 mg/kg/day to about50.00 mg/kg/day Preferred Range About 0.1 to about 5.0 mg/kg/day PTDCRange About 0.001 mg/kg/day to about 20.00 mg/kg/day Forskolin RangeAbout 0.001 mg/kg/day to about 20.00 mg/kg/day Preferred Range About0.01 to about 1.0 mg/kg/day Rolipram Range About 0.001 mg/kg/day toabout 20.00 mg/kg/day Preferred Range About 0.01 to about 2.0 mg/kg/dayMore Preferred About 0.1 to about 0.7 mg/kg/day Range Even More About0.5 mg/kg/day Preferred Range cAMP Range About 0.001 mg/kg/day to about20.00 mg/kg/day 8-bromo-cAMP Range About 0.001 mg/kg/day to about 20.00mg/kg/day FTP inhibitor II Range About 0.001 mg/kg/day to about 20.00mg/kg/day Preferred Range About 0.1 to about 20.0 mg/kg/day MorePreferred About 0.5 to about 10.0 mg/kg/day Range H-89 Range About 0.001mg/kg/day to about 20.00 mg/kg/day Myristoylated PKI More PreferredAbout 0.001 mg/kg/day to about Range 20.00 mg/kg/day (R)-cAMP MorePreferred About 0.001 mg/kg/day to about Range 20.00 mg/kg/day (S)-cAMPRange About 0.001 mg/kg/day to about 20.00 mg/kg/day 4-phenylbutyrateRange About 0.001 mg/kg/day to about (4PBA) 20.00 mg/kg/day 5-amino-Range About 0.001 mg/kg/day to about immidazole- 20.00 mg/kg/day4-carboxamide ribonucleoside (AICAR) theophylline Range About 0.001mg/kg/day to about 20.00 mg/kg/day papaverine Range About 0.001mg/kg/day to about 20.00 mg/kg/day

The pharmaceutical compositions disclosed herein may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or they may be compressed into tablets, or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compounds may be incorporated with excipientsand used in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. It will be understood to one of skill in the art that theactual amount of active ingredient used may vary depending on a numberof variables such as the symptoms of the patient, the size of thepatient and the age of the patient.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

The active compounds may also be administered parenterally e.g.intraperitoneally or intravascularly. Solutions of the active compoundsas free base or pharmacologically acceptable salts can be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention; of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral prophylaxis the polypeptide may be incorporated with excipientsand used in the form of non-ingestible mouthwashes and dentifrices. Amouthwash may be prepared incorporating the active ingredient in therequired amount in an appropriate solvent, such as a sodium boratesolution (Dobell's Solution). Alternatively, the active ingredient maybe incorporated into an antiseptic wash containing sodium borate,glycerin and potassium bicarbonate. The active ingredient may also bedispersed in dentifrices, including: gels, pastes, powders and slurries.The active ingredient may be added in a therapeutically effective amountto a paste dentifrice that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or other untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form.Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Therapeutic Kits

In one aspect, the invention provides a therapeutic kit comprising, insuitable container means, a therapeutically-effective amount of one ormore iNOS inhibitors and/or proinflammatory cytokine inhibitors and/orinduction suppressors selected from the group consisting of lovastatin,mevastatin, FPT inhibitor II, forskolin, rolipram, phenylacetate (NaPA),N-acetyl cysteine (NAC), PDTC, 4-phenylbutyrate (4PBA),5-aminoimmidazole-4-carboxamide ribonucleoside (AICAR), theophylline,papaverine, cAMP, 8-bromo-cAMP, (S)-cAMP, and salts, analogs, orderivatives therefrom, and if desired, a pharmaceutically acceptableexcipient. The compositions may be formulated such that they aresuitable for oral or parenteral administration.

In another aspect, the invention provides a therapeutic kit comprising,in suitable container means, a therapeutically-effective amount of oneor more iNOS inhibitors and/or proinflammatory cytokine inhibitorsand/or induction suppressors selected from agents that have certaintraits or modes of action common to those of the suppressors and/orinhibitors identified herein. Preferred substances would either inhibitthe Ras/Raf/MAP kinase pathway, inhibit and/or suppress the inductionand/or activation of NF-kB, inhibit mevalonate synthesis, be an enhancerof protein kinase A, and/or inhibit the farnasylation of proteins,including but not limited to Ras. In certain embodiments the inhibitorof mevalonate synthesis may be an inhibitor of HMG-CoA reductase orsuppressor of its induction. In certain aspects the inhibitor of HMG-CoAreductase is a stimulator of AMP-activated protein kinase. In certainother embodiments the inhibitor of inducible nitric oxide synthaseand/or proinflammatory cytokines may be an inhibitor of mevalonatepyrophosphate decarboxylase or suppressor of its induction. In otherembodiments the substance is an antioxidant. In other embodiments thesubstance is an enhancer of intracellular cAMP. The enhancer ofintracellular cAMP may be an inhibitor of cAMP phosphodiesterase and/orsuppressor of its induction. In other embodiments the substance is afarnesyl protein transferase inhibitor and/or induction suppressor.

In other preferred embodiments, a preferred stimulators or enhancerswould include a PKA inhibitor.

The other preferred embodiments, the inhibitors, suppressors stimulatorsor enhancers would be identified by the screening assay describedherein.

The diagnostic/therapeutic kits comprising the pharmaceuticalcompositions disclosed herein will generally contain, in suitablecontainer means, a therapeutically-effective amount of an iNOS and/orproinflammatory cytokine inhibitor and/or induction suppressor in apharmaceutically acceptable excipient. The kit may have a singlecontainer means that contains the iNOS and/or proinflammatory cytokineinhibitor and/or induction suppressor and a suitable excipient or it mayhave distinct container means for each compound.

The components of the kit may be provided as liquid solution(s), or asdried powder(s). When the components are provided in a liquid solution,the liquid solution is an aqueous solution, with a sterile aqueoussolution being particularly preferred. When reagents or components areprovided as a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. It is envisioned that the solvent mayalso be provided in another container means.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The iNOS and/orproinflammatory cytokine inhibitor(s) and/or induction suppressor(s) mayalso be formulated into a syringeable composition. In which case, thecontainer means may itself be a syringe, or other such like apparatus,from which the formulation may be administered into the body, preferablyby injection or even mixed with the other components of the kit prior toinjection. The iNOS and/or proinflammatory inhibitor and/or inductionsuppressor to be administered may be a single inhibitor, or acomposition comprising two or more inhibitors in a single or multipledose for administration. Alternatively, one or more inhibitors may beadministered consecutively or concurrently with other agents as deemedappropriate by the clinician. Dosage of each of the compositions willvary from subject to subject depending upon severity of conditions,size, body weight, etc. The calculation and adjustment of dosages ofpharmaceutical compositions is well-known to those of skill in the art.

In an alternate embodiment, components of the kit may be provided asdried powder(s). When reagents or components are provided as a drypowder, the powder can be reconstituted by the addition of a suitablesolvent. It is envisioned that the solvent may also be provided inanother container means.

The container means will generally include at least one vial, test tube,flask, bottle, syringe or other container means, into which the iNOSand/or proinflammatory cytokin inhibitor and/or induction suppressor maybe placed, preferably, suitably allocated. Where two or more inhibitorsand/or supressors are provided, the kit will also generally contain asecond vial or other container into which this additional inhibitorsand/or suppressors may be formulated. The kits may also comprise asecond/third container means for containing a sterile, pharmaceuticallyacceptable buffer or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection or blow-molded plastic containers into which thedesired vials are retained. Alternatively, the vials may be prepared insuch a way as to permit direct introduction of the composition into anintravenous drug delivery system.

Irrespective of the number or type of containers, the kits of theinvention may also comprise, or be packaged with, an instrument forassisting with the injection/administration or placement of the ultimateiNOS and/or proinflammatory cytokine inhibitor and/or inductionsuppressor composition within the body of an animal. Such an instrumentmay be a syringe, pipette, forceps, measured spoon, eye dropper or anysuch medically approved delivery vehicle.

The term “iNOS and/or proinflammatory cytokine inhibitor and/orinduction suppressor” and also includes derivatives of the compoundsdisclosed herein which exhibit at least some biological activity incommon with the unmodified compound. In general these compounds areinhibitors of inducible nitric oxide synthase and/or proinflammatorycytokines.

The following examples are included to demonstrate new and inventivemethods of the inventor and preferred embodiments of the invention. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the examples which follow represent techniques discoveredby the inventor to function well in the practice of the invention, andthus can be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

EXAMPLE 1 Inhibition of iNOS and Cytokines

Reagent Recombinant rat IFN-γ, DMEM/F-12 medium, RPMI 1640 medium, fetalbovine serum and Hanks' balanced salt solution (HBSS) were obtained fromGIBCO. Human IL1-β was obtained from Genzyme, USA. Mouse recombinantTNF-α was obtained from Boehringer Mannheim, Germany. Lovastatin,mevastatin and farnesyl pyrophosphate were obtained from Biomol, USA andCalBiochem, USA. Cholesterol, ubiquinone, arginase, N-Acetyl cystein(NAC), pyrrolidine dithiocarbamate (PDTC), NADPH, FAD,tetrahydrobiopterin, Dowen-50W and LPS (Escherichia coli) were obtainedfrom Sigma (St. Louis, Mo.). N^(G)-methyl-L-arginine (L-NMA), FPTinhibitor II and antibodies against mouse macrophage iNOS were obtainedfrom Calbiochem, USA. Immunoassay kits for TNF-α, IL-1β and IL-6 wereobtained from R&D, USA. NF-kβ DNA binding protein detection kit wasobtained from GIBCO/BRL, USA. [γ-³²P]ATP (3000 Ci/mmol) andL-[2,3,4,5-³H] arginine were obtained from Amersham, USA. Sodium salt ofphenylacetic acid (NaPA) was prepared from phenylacetic acid (Sigma) andNaOH as described (Samid et al., 1992).

Induction of NO Production in Rat Astrocytes, Microglia and C6 GlialCells Astrocytes were prepared from rat cerebral tissue as described byMcCarthy et al. (1980). Cells were maintained in DMEM/F-12 mediumcontaining 10% fetal bovine serum (FBS). After 10 days in culture,astrocytes were separate from microglia and oligodendrocytes by shakingfor 24 h in an orbital shaker at 240 rpm. To ensure the complete removalof all oligodendrocytes and microglia, the shaking was repeated twiceafter a gap of one or two days before subculturing. The microglialcontamination was checked by non-specific esterase staining andoligodendrocytes were examined by immunofluorescence using antibodiesagainst GC (McCarthy et al., 1980). Cells were trypsinized, subculturedand stimulated with LPS or different cytokines in serum-free DMEM/F-12medium.

Microglial cells were isolated from mixed glial cultures according tothe procedure of Guilian et al. (1986). Briefly, on day 7 to 9 the mixedglial cultures were washed 3 times with DMEM/F-12 and subjected to ashake at 240 rpm for 2 h at 37° C. on a rotary shaker. The floatingcells were washed and seeded on to plastic tissue culture flasks andincubated at 37° C. for 2 h. The attached cells were removed bytrypsinization and seeded on to new plates for further studies. Ninetyto ninety-five percent of this preparation was found to be positive fornon-specific esterase, a marker for macrophages and microglia. For theinduction of NO production, cells were stimulated with LPS or cytokinesin serum-free condition.

C6 glial cells, obtained from ATCC, were also maintained and inducedwith different stimuli as indicated above.

Treatment of Cells With iNOS Inhibitors Cells in culture were treatedwith these compounds by addition of these compounds to the cell culturemedia. Dose ranges are provided in the accompanying figures and tables.

Isolation of Rat Macrophages and Induction of NO Production Residentmacrophages were obtained from rats by peritoneal lavage with sterileRPMI 1640 medium containing 1% fetal bovine serum and 100 μg/mlgentamicin (Wang et al., 1995). Cells were washed three times with RPMI1640 at 4° C. All cell cultures were maintained at 37° C. in ahumidified incubator containing 5% CO₂ in air. Macrophages, at aconcentration of 2×10⁶/ml in RPMI 1640 medium containing L-glutamine andgentamicin, were added in volumes of 800 μl to a 35 mm plate. After 1 h,nonadherent cells were removed by washing and 800 μl of serum-free RPMI1640 medium with various stimuli were added to the adherent cells. After24 h incubation in 5% CO₂ in air at 37° C., the culture supernatantswere transferred to measure NO production (Geng et al., 1995; Wang etal., 1995).

Assay of the Viability of Cells Treated with Lovastatin, Mevastatin,NaPA, TNF-α, IL-1β, IFN-γ, or LPS The cytotoxic effects of the compoundsused in various studies disclosed herein were determined by measuringthe cell viability by trypan blue exclusion.

It was found that none of the compounds lovastatin, mevastatin, NaPA,TNF-α, IL-1β, IFN-γ, or LPS had a significant effect on the viability ofastrocytes, microglia or macrophages. Changes in cell viability cantherefore be ruled out as a cause for the disclosed findings.

Assays of NO synthesis and NOS Activity Synthesis of NO was determinedby assay of culture supernatants for nitrite, a stable reaction productof NO with molecular oxygen. Briefly, 400 μl of culture supernatant wasallowed to react with 200 μl of Griess reagent (Feinstein et al., 1994a;Wang et al., 1995) and incubated at room temperature for 15 min. Theoptical density of the assay samples was measured spectrophotometricallyat 570 nm. Fresh culture media served as the blank in all experiments.Nitrite concentrations were calculated from a standard curve derivedfrom the reaction of NaNO₂ in the assay. Protein was measured by theprocedure of Bradford (1976).

NOS activity was measured directly by production ofL-[2,3,4,5-³H]citrulline from L-[2,3,4,5-³H] arginine (Feinstein et al.,1994a). In these studies, 50 μl of macrophage homogenate was incubatedat 37° C. in presence of 50 mM Tris-HCl (pH 7.8), 0.5 mM NADPH, 5 μMFAD, 5 μM tetrahydrobiopterin and 12 μM L-[2,3,4,5-³H]arginine (118mCi/mmol) in a total volume of 200 μl. Assays were carried out for 30 to40 min and the production of L-[2,3,4,5-³H] citrulline was linear. Thereactions were stopped by addition of 800 μl of ice-cold 20 mM HEPES (pH5.5) followed by addition of 2 ml of Dowex-50W equilibrated in the samebuffer. The samples were then centrifuged and the concentration ofL-[³H]citrulline was determined in the supernatant by liquidscintillation counting. Protein was measured by the procedure ofBradford (1976).

Immunoblot analysis for iNOS Following 24 h incubation in the presenceor absence of stimuli by different cytokines or LPS, macrophages werescraped off, washed with Hank's buffer, and homogenized in 50 mMTris-HCl (pH 7.4) containing protease inhibitors (1 nM PMSF, 5 μg/mlaprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin). Afterelectrophoresis the proteins were transferred onto a nitrocellulosemembrane, and the iNOS band was visualized by immunoblotting withantibodies against mouse macrophage iNOS and [¹²⁵I]-labeled protein A(Singh et al., 1988).

Cells pre-incubated in serum-free media with different concentrations oflovastatin (5 or 10 μM) or NaPA (2 or 5 mM) or a combination of 2 μMlovastatin and 2 mM NaPA for 8 h received 1.0 μg/ml of LPS. Cellhomogenates were electrophoresed, transferred on nitrocellulose membraneand immunoblotted with antibodies against mouse macrophage iNOS asdescribed in Example 7. Western blot analysis for iNOS protein ofLPS-stimulated astrocytes clearly showed that both lovastatin and NaPAsignificantly inhibited the LPS-mediated induction of iNOS protein. Acombination of lovastatin and NaPA at dose lower than the one usedindividually almost completely inhibited LPS-induced production of NOand expression of iNOS.

RNA isolation, Northern blot analysis, and reverse-transcriptase coupledpolymerase chain reaction (RT-PCR) Stimulated peritoneal macrophageswere taken out from culture dishes directly by adding Ultraspec-II RNAreagent (Biotecx Laboratories Inc.) and total RNA was isolated accordingto the manufacturer's protocol. For northern blot analyses, 20 μg oftotal RNA was electrophoresed on 1.2% denaturing formaldehyde-agarosegels, electrotransferred to Hybond-Nylon Membrane (Amersham) andhybridized at 68° C. with ³²P-labeled cDNA probe using Express Hybhybridization solution (Clontech) as described by the manufacturer. ThecDNA probe was made by PCR™ amplification using two primers (forwardprimer: 5′-CTCCTTCAAAGAGGCAAAAATA-3′ (SEQ ID NO:1); reverse primer:5′-CACTTCCTCCAGGATGTTGT-3′ (SEQ ID NO:2)) (Geller et al., 1993). Afterhybridization filters were washed two to three times in solution I(2×SSC, 0.05% SDS) for 1 h at room temperature followed by solution II(0.1×SSC, 0.1% SDS) at 50° C. for another hour. The membranes were thendried and exposed to X-ray film (Kodak). The same filters were strippedand rehybridized with probes for GAPDH. The relative mRNA content foriNOS was measured after scanning the bands with a Biorad (Model GS-670)imaging densitometer.

Five micrograms of total RNA was reverse transcribed by using oligo-dTby using 1 mM of each dNTP, 40 U of RNase inhibitor (Promega), 50 U ofMoloney murine leukemia virus (M-MLV) reverse transcriptase (Stratagene)and reverse transcription buffer (Stratagene) in a 50 μl reactionvolume. The integrity of the RNAs was checked by running an alkaline RNAgel. The first strand cDNA synthesis was carried out at 37° C. for 1 h.To check the quality of cDNAs for iNOS (730 bp) and for GAPDH (528 bp),the same cDNA was used as a control to amplify 1.5 kb fragment of a iNOSgene. Five microliters of this 1st strand cDNA was used to amplify byPCR™ in 100 μl reaction volume containing 0.2 μM of each primer, 200 μMof each dNTP, manufacturer-supplied 1× buffer containing 1.5 mM MgCl₂and 2.5 U of Taq DNA polymerase (Stratagene). A total of 30 cycles wererun with each cycle having denaturation at 91° C. for 1 min, annealingat 54° C. for 1 min and extension at 72° C. for 2 min. Final extensionwas carried out 72° C. for 10 min. Oligonucleotide primers for iNOS(forward primer: 5′-CTCCTTCAAAGAGGCAAAAATA-3′ (SEQ ID NO:1); reverseprimer: 5′-CACTTCCTCCAGGATTGGTG-3′ (SEQ ID NO:3)) were synthesized basedon the sequences described by Geller et al. (1993). The PCR™ amplifiedproducts were flirter confirmed by restriction mapping. Oligonucleotideprimers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forwardprimer: 5′-ACCACCATGGAGAAGGCTGG-3′ (SEQ ID NO:4); reverse primer:5′-CTCAGTGTAGCCCAGGAT GC-3′ (SEQ ID NO:5)) were used as control. PCR™products were visualized by electrophoresis in 1.5% agarose gelcontaining 0.5 μg/ml ethidium bromide and photographed with a DS-34 typecamera. The relative mRNA content for iNOS was measured after scanningthe bands with a Biorad (Model GS-670) imaging densitometer.

After 5 h of incubation, cells were taken out directly by addingultraspec-II RNA reagent (Biotecx Laboratories Inc., Houston, Tex.) tothe plates for isolation of total RNA, and northern blot analysis foriNOS mRNA was carried out as described in Example 7. The Northern blotanalysis for iNOS mRNA of LPS-stimulated astrocytes clearly showed thatboth lovastatin (5 or 10 μM) and NaPA (2 or 5 mM) significantlyinhibited the LPS-mediated induction of iNOS mRNA. A combination oflovastatin and NaPA, at 2 μM and 2 mM respectively, at a dose lower thanthe one used individually almost completely inhibited LPS-inducedproduction of NO and expression of iNOS.

Determination of TNF-α, IL-1β and IL-6 in culture supernatantsMacrophages were stimulated with LPS and IFN-γ in serum-free RPMI 1640media for 24 h in the presence or absence of NAC, PDTC, lovastatin orNaPA, and concentrations of TNF-α, IL-1β and IL-6 were measured inculture supernatants by using a high-sensitivity enzyme-linkedimmunosorbent assay (ELISA; Genzyme, Cambridge, Mass.; R&D Systems, USA)according to the manufacturer's instructions.

Preparation of Nuclear Extracts and Electrophoretic Mobility shift assayNuclear extracts from stimulated or unstimulated astrocytes (1×10⁷cells) were prepared using the method of Dignam et al. (1983) withslight modification. Cells were harvested, washed twice with ice-coldphosphate-buffered saline and lysed in 400 μl of buffer A (10 mM HEPES,pH 7.9, 10 mM KCl, 2 mM MgCl₂, 0.5 mM DTT, 1 mM PMSF, 5 μg/ml aprotinin,5 μg/ml pepstatin A, and 5 μg/ml leupeptin (Sigma, St. Louis, Mo.)containing 0.1% Nondet P-40 for 15 min on ice, vortexed vigorously for15 s, and centrifuged at 14,000 rpm for 30 s. The pelleted nuclei wereresuspended in 40 μl of buffer B (20 mM HEPES, pH 7.9, 25% (v/v)glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF,5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin). After 30min on ice, lysates were centrifuged at 14,000 rpm for 10 min.Supernatants containing the nuclear proteins were diluted with 20 μl ofmodified buffer C (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.05 M KCl,0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF) and stored at −70° C. untiluse. Nuclear extracts were used for the electrophoretic mobility shiftassay using the NF-kβ DNA binding protein detection system kit(GIBCO/BRL), according to the manufacturer's protocol.

Inhibition Of LPS- And Cytokine-Induced Production Of NO By LovastatinTo examine whether cytokine-induced NO production is inhibited bylovastatin, rat primary astrocytes were stimulated with differentcombinations of LPS, TNF-α, IL-1β and IFN-γ (i.e. LPS+TNF-α, LPS+IFN-γ,TNF-α+IL-1β, TNF-α+IFN-γ) for 24 h and the production of NO was measuredas described above. Cells, pre-incubated in serum-free media with 10 μMlovastatin for 8 h, received different combinations of LPS andcytokines. Concentration of different stimuli were: LPS, 0.5 μg/ml;TNF-α, 20 ng/ml; IL-1β, 50 ng/ml; IFN-γ, 50 U/ml. After 24 h ofincubation, the production of nitrite was measured in supernatants. Datawas taken as the mean±S.D. of three different studies. All thecombinations of LPS and cytokines significantly induced the productionof NO, however, the addition of 10 μM lovastatin to astrocytes inhibitedthe NO production and induction of iNOS protein. Cell homogenates wereanalyzed for iNOS protein by immunoblotting as described. Under similarconditions lovastatin was also found to inhibit LPS- andcytokine-induced NO production in rat C6 glial cells.

Inhibition of LPS-Induced Expression of iNOS by Lovastatin, NaPA andMevastatin An examination was made of the effect of lovastatin,mevastatin and mevalonate pyrophosphate decarboxylase (NaPA) on theinduction of iNOS and production of NO. As shown in Table 1, it wasfound that bacterial LPS at a concentration of 1.0 μg/ml induced theproduction of NO by about 8-fold. The inhibition of NO production byarginase, an enzyme that degrades the substrate (L-arginine) of NOS andL-NMA, a competitive inhibitor of NOS, indicate that LPS-induced NOproduction in astrocytes is dependent on NOS-mediated argininemetabolism (Table 2). Lovastatin or mevastatin alone was neitherstimulatory nor inhibitory to nitrite production in control astrocytes.However, both the inhibitors, when added 8 h before the addition of LPS,inhibited LPS-mediated induction of nitrite production in astrocytes.Only 25% inhibition in LPS-induced NO production was found whenlovastatin was added to the cells along with LPS; however, the degree ofinhibition increased with the increase in time of preincubation withlovastatin reaching about 90% inhibition of NO production within 8 to 10h of preincubation. Lovastatin (5 or 10 μM) or NaPA (2 or 5 mM) or acombination of 2 μM lovastatin and 2 mM NaPA also inhibited theinduction of NO production in rat primary astrocytes. After 24 h,supernatants were used for nitrite assay as described above. Data wasthe mean±S.D. of three different studies.

TABLE 2 Inhibition of LPS-induced NO production in rat primaryastrocytes by lovastatin and mevastatin Stimuli Nitrite (nmol/mg/24 h) %Inhibition Control 2.9 ± 0.5 — LPS 25.3 ± 3.2  — LPS + Arginase 5.9 ±0.8 87 LPS + L-NMA 5.5 ± 0.7 88 Lovastatin 2.9 ± 0.3 — Mevastatin 2.8 ±0.4 — LPS + Lovastatin 5.2 ± 0.5 90 LPS + Mevastatin 5.5 ± 0.5 88Astrocytes were cultured for 24 h in serum-free DMEM/F-12 with thelisted reagents; and nitrite concentration in the supernatants weremeasured as described. Arginase (100 units/ml) and L-NMA (0.1 mM) wereadded to the cells together with LPS (1.0 μg/ml). Data are mean±standarddeviation (S.D.) of three different studies.

Inhibition of LPS-Induced Activation of NF-kβ and Expression of iNOS byLovastatin and NaPA The effect of lovastatin (5 or 10 μM) or NaPA (2 or5 mM) on LPS-induced activation of NF-kβ in astrocytes was examined bygel-shift DNA-binding assay. Cells incubated in serum-free mediareceived 1.0 μg/ml of LPS. After 1 h of incubation, cells were taken outto prepare nuclear extracts and nuclear proteins were used for theelectrophoretic mobility shift assay of NF-kβ as described in Example10. Lanes were run containing control, LPS, LPS-treated nuclear extractwith 25-fold excess of unlabelled probe, and LPS-treated nuclear extractwith 50-fold excess of unlabelled probe. Treatment of rat primaryastrocytes with 1.0 μg/ml of LPS resulted in the activation of NF-kβ.This gel shift assay detected a specific band in response to LPS thatwas competitively removed by an unlabelled probe. Lovastatin or NaPAalone at different concentrations failed to induce NF-kβ. However, cellspreincubated in serum-free media with lovastatin or NaPA for 8 h weremarkedly inhibited for the LPS-induced activation of NF-kβ, indicatingthat the inhibition of iNOS expression by lovastatin and NaPA is due tothe inhibition of NF-kβ.

To evaluate the possible mechanism of the effect of lovastatin and NaPAor to determine whether reduced concentrations of end products asopposed to intermediate products of the mevalonate pathway wereresponsible for the effects of lovastatin and NaPA, the inventorperformed rescue experiments with cholesterol, ubiquinone, mevalonateand farnesyl pyrophosphate (FPP). Cells preincubated in serum-free mediawith 10 μM of lovastatin or 5 mM of NaPA for 8 h received 1.0 μg/ml ofLPS along with 100 μM mevalonate or 200 μM farnesyl pyrophosphate. After24 h, supernatants were used for a nitrite assay as described in Example6. Combinations that were tested included control, LPS alone,LPS+lovastatin, LPS+lovastatin+mevalonate, LPS+lovastatin+FPP, LPS+NaPA,LPS+NaPA+mevalonate, and LPS+NaPA+FPP. Data was measured as themean±S.D. of three different studies. After 5 h of incubation, cellswere analyzed for iNOS mRNA by northern blotting technique as described.GAPDH mRNA was also measured. After 1 h of incubation, cells were takenout to prepare nuclear extracts and nuclear proteins were used for theelectrophoretic mobility shift assay of NF-kβ as described in Example10. Addition of 10 μM ubiquinone or cholesterol to astrocytes did notprevent the inhibitory effect of lovastatin and NaPA. These observationssupport the conclusion that the depletion of intermediary productsrather than end products of mevalonate pathway are responsible for theobserved inhibitory effect of lovastatin or NaPA on LPS-induced iNOSexpression. On the other hand, mevalonate of FPP substantially reversedthe inhibitory effect of lovastatin on iNOS expression and NF-kβactivation. However, FPP not mevalonate reversed the inhibitory effectof NaPA indicating that the utilization of mevalonate rather than itssynthesis is the prime target of the NaPA.

Inhibition of LPS-Induced Expression of iNOS in Rat Primary Astrocytesby FPT inhibitor II An examination was made of the effect of FPTinhibitor II, an inhibitor of enzymes that transfers farnasyl group toproteins (e.g. Ras), on LPS-mediated expression of iNOS and activationof NF-kβ in rat primary astrocytes. Cells pre-incubated in serum-freemedia with 100 μM or 200 μM FPT inhibitor II for 1 h received 1.0 μg/mlof LPS. Samples assayed included control, LPS, LPS+FPT inhibitor II (100μM or 200 μM). After 24 h of incubation, supernatants were used fornitrite assay as described in Example 6. Data was measured as themean±S.D. of three different studies. After 5 h of incubation, cellswere analyzed for iNOS mRNA by northern blotting technique as described.After 1 h of incubation, cells were taken out to prepare nuclearextracts and nuclear proteins were used for the electrophoretic mobilityshift assay of NF-kβ as described in Example 10. A preincubation ofcells for 1 h with 100 or 200 μM FPT inhibitor II inhibited LPS-inducedactivation of NF-kβ, expression of iNOS and production of NO; thus,demonstrating the importance of farnesylation of Ras in LPS-mediatedactivation of NF-kβ and induction of iNOS in astrocytes.

Lovastatin and NaPA inhibit the LPS-induced expression of Cytokines Anexamination was made of the effect of NaPA and lovastatin on LPS-inducedexpression of TNF-α, IL-1β and IL-6. Rat primary astrocytespre-incubated in serum-free media with different concentrations oflovastatin (5 or 10 μM) or NaPA (2 or 5 mM) or a combination of 2 μM oflovastatin and 2 mM of NaPA for 8 h received 1.0 μg/ml of LPS.Combinations that were tested included control, LPS, LPS+lovastatin (5μM), LPS+lovastatin (10 μM), NaPA (2 μM), LPS+NaPA (5 μM), andLPS+lovastatin (2 μM)+NaPA (2 μM). Concentrations of TNF-α, IL-1β andIL-6 were measured in the supernatants after 24 h of incubation (Table3) and the mRNA expression of these cytokines was examined in the cellsafter 5 h of LPS stimulation as described. Bacterial LPS markedlyinduced the mRNA expression and production of respective cytokines inastrocytes. Although lovastatin or NaPA alone had no effect on theproduction of cytokines, these two compounds strongly inhibited theLPS-induced production of TNF-α, IL-1β and IL-6 in the supernatants(Table 3). Additionally, 2 mM NaPA and 2 μM lovastatin worked moreeffectively to inhibit LPS-induced production of TNF-α, IL-1β and IL-6than 5 mM NaPA or 5 μM lovastatin alone. The decrease in cytokineproduction was also accompanied by an inhibition of their mRNAexpression demonstrating that lovastatin and NaPA down-regulate theexpression of all the inflammatory mediators (iNOS, TNF-α, IL-1β andIL-6) in astrocytes. No adverse effects on the viability of astrocytes,as measured by trypan blue exclusion, were observed.

TABLE 3 Inhibition of LPS-induced production of NO, TNF-α, IL-1β andIL-6 in rat primary astrocytes, microglia and macrophages by lovastatinand NaPA Production of Treatments NO or LPS + Cells cytokines LPS onlyLovastatin LPS + NaPA Astrocytes NO 25.3 ± 3.2 5.2 ± 0.4 5.4 ± 0.6 TNF-α 5.3 ± 0.8  0.3 ± 0.05  0.4 ± 0.06 IL-1β 10.4 ± 1.5 0.8 ± 0.1 1.1 ± 0.2IL-6 136.5 ± 16.8 6.9 ± 0.9 7.6 ± 0.8 Microglia NO 81.2 ± 6.9 5.9 ± 0.46.9 ± 0.9 TNF-α 14.5 ± 2.1 0.9 ± 0.1 1.3 ± 0.2 IL-1β 28.2 ± 3.4 2.1 ±0.3 2.4 ± 0.2 IL-6 295.6 ± 33.5 7.8 ± 1.1 9.3 ± 1.2 Macrophages NO 118.5± 12.5 7.2 ± 0.9 9.5 ± 0.7 TNF-α 18.6 ± 2.3 1.2 ± 0.1 1.7 ± 0.2 IL-1β34.6 ± 4.5 2.3 ± 0.3 3.1 ± 0.4 IL-6 350.0 ± 27.6 8.3 ± 0.6 10.2 ± 1.4 Cells preincubated with 10 μM lovastatin or 5 mM NaPA for 8 h inserum-free condition was stimulated with 1.0 μg/ml of LPS. After 24 h ofincubation, concentrations of NO, TNF-α, IL-1β and IL-6 were measured insupernatants as described above. NO is expressed as nmol/24 h/mg proteinwhereas TNF-α, IL-1β and IL-6 are expressed as ng/24 h/mg protein. Dataare expressed as the mean±S.D. of three different experiments.

Inhibition Of LPS-Induced Production Of NO and Cytokines In Rat PrimaryMicroglia And Macrophages By Lovastatin Both macrophages and microglia,important sources of NO and cytokines, actively participate in thepathophysiologies of different inflammatory disorders. Since lovastatinand NaPA inhibited the LPS-induced production of NO, TNF-α, IL-1β andIL-6 in astrocytes, a determination of the effect of lovastatin and NaPAon LPS-stimulated production of NO, TNF-α, IL-1β and IL6 in rat primarymicroglia and macrophages was made (Table 3). It was found that the rateof production of NO and cytokines after LPS stimulation was much higherin both macrophages and microglia than in astrocytes. Similar toastrocytes, lovastatin or NaPA alone had no effect on the production ofNO and cytokines in macrophages and microglia. However, both of thesecompounds strongly inhibited the LPS-induced production of NO, TNF-α,IL-1β and IL-6 in macrophages and microglia (Table 3). These resultsdemonstrate the importance of these compounds in controlling iNOSproduced NO and production of proinflammatory cytokines (TNF-α, IL-1βand IL-6 and IFN-γ) in microglia and macrophages. It is important tonote that under the conditions used, no adverse effects on the viabilityof microglia or macrophages, as measured by trypan blue exclusion, wereobserved.

EXAMPLE 2 NAC Inhibits LPS-Induced NO Synthesis in Resident PeritonealMacrophages

Reagents Reagents were as given in Example 1. Rat macrophages,astrocytes and C6 glial cells were prepared as described in Example 1.Assays to measure the induction of NO synthesis, NOS activity,immunoblot analyses for iNOS, RNA isolation, RT-PCR and determination ofTNF-α in culture supernatants were as described in Example 1.

The effect of NAC on LPS-induced NO-synthesis was examined in residentperitoneal macrophages. Resident macrophages were cultured in RPMImedium without serum in presence of different concentrations of LPS andNAC. The concentration of NO as nitrite was measured in culturedsupernatants after 24 h. As shown in Table 4, in rat residentmacrophages, LPS (1 μg/ml) induced the production of nitrite, thesoluble product of NO in the culture medium (Wang et al., 1995), by morethan ten fold. LPS-induced production of nitrite was concentrationdependent with maximal induction at 1-5 μg/ml of LPS (data not shown).NAC itself was neither stimulatory nor much inhibitory to nitriteproduction in control resident macrophages. However, NAC, when added 2 hbefore the addition of LPS, inhibited LPS-mediated induction of nitriteproduction in macrophages. Over 90% inhibition was observed when NAC wasused at a concentration of 20 mM. Both L-NMA, a competitive inhibitor ofNOS, and arginase suppressed LPS-mediated nitrite secretion, indicatingthat LPS-induced nitrite release in rat peritoneal macrophages isdependent on NOS-mediated arginine metabolism (Table 4).

TABLE 4 Inhibition of Arginine-Dependent Nitrite Accumulation inLPS-Stimulated Resident Macrophages by NAC Nitrite Stimuli (nmol/mgProtein^(24h)) Inhibition (%) Control  9.8 ± 1.5 NAC  6.2 ± 0.8 lps124.2 ± 9.7  LPS + NAC 11.6 ± 1.5 91 LPS + NMA 15.8 ± 2.2 87 LPS +arginase 22.5 ± 3.1 82Resident macrophages were cultured for 24 h in serum-free RPMI 1640 withthe listed reagents; nitrite concentration in the supernatants was thenmeasured as described in the methods section. Concentration of reagentswere: LPS, 1.0 μg/ml; NAC, 20 mM; NMA, 0.1 mM; arginase, 100 units/ml.NMA and arginase were added to the cells together with LPS whereas NACwas added 2 h before the addition of LPS. Data are mean±S.D. of threedifferent experiments.

Kinetics of inhibition of NO synthesis by NAC in rat macrophages Todetermine whether inhibition of LPS-induced NO synthesis by NAC wassimply due to delayed induction, nitrite concentrations were measured inLPS-stimulated cultures maintained up to 48 h. Rat resident macrophageswere stimulated with 1.0 μg/ml LPS alone or together with 20 mM NAC,where NAC was added 2 h before the addition of LPS. Supernatants wereharvested at different time intervals (6 to 48 h) to measureconcentrations of nitrite as described. Data was measured as themean±S.D. of three different studies. When cells were stimulated in theabsence of NAC, nitrite was detected in culture supernatants after 8 hand the concentration of nitrite increased progressively thereafter for48 h. However, when 20 mM NAC was added 2 h before the addition of LPS,nitrite production was significantly inhibited. From the onset ofdetectable NO release until 24 h, nitrite accumulated at a rate of 5.2nmol/mg/h in the absence of NAC and at 0.5 nmol/mg/h in the presence ofNAC.

To study the Effect of decreasing or increasing the time intervalbetween addition of LPS and NAC on LPS-stimulated macrophage NOproduction, resident macrophages were incubated with 1.0 μg/ml LPS. NAC(20 mM) was added to cultures 2 h or 1 h before, simultaneously or 1 h,3 h, 5 h, or 7 h after the addition of LPS. Supernatants were collected24 h after the addition of LPS. Each value was determined as themean±S.D. of three different studies. Culture supernatants werecollected after 24 h of incubation to measure the concentration ofnitrite. Maximal suppression of nitrite production was observed when NACwas added 2 h before the addition of LPS. When NAC was added after theaddition of LPS, the extent of inhibition progressively decreased. Only20% inhibition was observed when NAC was added 7 h after the addition ofLPS, indicating that inhibition of nitrite production by NAC is due tothe inhibition of oxygen radical-mediated signaling reactions.

NAC and PDTC inhibit LPS-mediated induction of iNOS in rat residentmacrophages To understand the mechanism of inhibition of LPS-inducednitrite production in resident macrophages by antioxidants, anexamination was made of the effect of NAC and PDTC on the formation ofL-citrulline from L-arginine, the reaction which is catalyzed by NOS inhomogenates of macrophages. Homogenates were prepared from macrophagesthat had been incubated for 24 h with 1.0 μg/ml LPS in the presence orabsence of 20 mM NAC. NAC was added to the cells 2 h before the additionof LPS. Production of L-[2,3,4,5-³H]citrulline fromL-[2,3,4,5³-H]arginine was determined at different time points. Data wasdetermined as the mean of two separate studies. The formation ofL-citrulline from L-arginine was linear up to 40 min in LPS-activatedmacrophages in the absence of NAC whereas in the presence of NAC,formation of L-citrulline was strongly inhibited.

In another study, cells received different concentrations of NAC andPDTC, 2 h before the addition of 1.0 μg/ml LPS. Samples that were testedincluded control, LPS, 5 mM NAC+LPS, 10 mM NAC+LPS, 20 mM NAC+LPS, 50 μMPDTC+LPS, and 100 μM PDTC+LPS. After 24 h of incubation, cells werewashed, scraped off, and homogenized. NOS activity was measured in cellhomogenates as described. Data was determined as the mean±S.D. of threedifferent studies. Cell homogenates were electrophoresed, transferred onnitrocellulose membrane, and immunoblotted with antibodies against mousemacrophage iNOS as described. After 6 h of incubation, cells were takenout from culture dishes directly by adding ultraspec-II RNA reagent(Biotecx Laboratories Inc.). Total RNA of each sample was prepared,reverse-transcribed and amplified by using specific primers for iNOS andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. PCR™ productswere electrophoresed in 1.5% agarose gel containing 0.5 μg/ml ethidiumbromide and photographed with a DS-34 type polaroid camera.

Both NAC and PDTC inhibited LPS-induced NOS activity as evidenced byL-citrulline formation, and PDTC at a concentration of 100 μM was aspotent as NAC at 20 mM. Immunoblot analysis with antibodies againstmurine macrophage iNOS and RT-PCR for iNOS mRNA analysis ofLPS-stimulated macrophages incubated in the presence or absence of NACor PDTC show that both the antioxidants inhibited LPS-mediated inductionof iNOS protein and mRNA, indicating that LPS induced induction of iNOSprotein in macrophages via oxygen radicals signal pathway.

Inhibition Of LPS- And/Or Cytokine-Induced NO Production By NACPeritoneal macrophage iNOS can be induced not only by LPS but also byIFN-γ, in combination with either IL-1β or TNF-α (Mehta et al., 1994).To determine whether cytokine-induced NO synthesis is also inhibited byNAC, resident macrophages were cultured with TNF-α, IL-1β or IFN-γseparately or in several combinations, in the presence or absence ofNAC. IL-1β or IFN-α when added alone was not able to induce nitriteproduction; whereas, IFN-γ alone significantly increased NOS-mediatednitrite production (Table 5). Additionally, different combinations ofcytokines and LPS induced high level of nitrite production and NOSactivity (Table 5). However, NAC, when added 2 h before the addition ofcytokines, potentially inhibited the induction of nitrite production.This inhibition of nitrite production was associated with the inhibitionof NOS activity as measured by the formation of L-citrulline (Table 5).

NAC and PDTC inhibit LPS/cytokines stimulated induction of iNOS in ratmacrophages Cells received 20 mM NAC 2 h before the addition ofdifferent cytokines. After 24 h of incubation with different cytokines,cells were scraped off, washed, and homogenized. Homogenates wereimmunoblotted with antibodies against mouse macrophage iNOS as describedin Example 7. After 6 h of incubation with different stimuli, cells weretaken out from culture dishes directly by adding ultraspec-II RNAreagent (Biotecx Laboratories Inc.). Total RNA of each sample wasprepared, reverse-transcribed, and amplified by using specific primersfor iNOS and GAPDH mRNA. PCR™ products were electrophoresed in 1.5%agarose gel containing 0.5 μg/ml ethidium bromide and photographed witha DS-34 type polaroid camera. Bands were scanned with a Biorad (ModelGS-670) imaging densitometer. The ratio of iNOS gene product to theinternal standard, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) wasused to quantitate the message level. Results were measured as themean±S.D. of three different studies. Assays were done for control,LPS+TNF-α, LPS+IL-1β, LPS+IFN-γ, TNF-α+IL-1β, TNF-α+IFN-γ,NAC+LPS+TNF-α, NAC+LPS+IL-1β, NAC+LPS+IFN-γ, NAC+TNF-α+IL-1β, andNAC+TNF-α+IFN-γ. Concentrations of different stimuli were 0.2 μg/ml forLPS, 50 U/ml for IFN-γ, 20 ng/ml for TNF-α, and 25 ng/ml for IL-1β. TheLPS and cytokine combinations stimulated nitrite production, while NACin combination with LPS and/or cytokines inhibited nitrite production.This inhibition was associated with the inhibition of NOS activity asmeasured as well as the inhibition of expression of the iNOS protein andmRNA, indicating with the results described above for Table 5 that LPS-and cytokines-mediated expression of iNOS involves oxygen radicalssignal pathway.

TABLE 5 Inhibition nitrite production and NOS activity by NAC instimulated rat resident macrophages NAC Nitrite L-citrulline (20 (nmol/Inhibition (pmol/ Inhibition Stimuli mM) mg/24 h) (%) min/mg) (%)Control −  9.8 ± 1.5 13.5 ± 1.8 +  6.2 ± 0.8 37 10.2 ± 0.9 25 LPS −124.2 ± 9.7  156.4 ± 21.2 + 11.6 ± 1.5 91 14.2 ± 2.3 91 IL-1β − 21.4 ±1.9 22.5 ± 3.2 +  9.2 ± 0.7 57 12.9 ± 2.1 43 TNF-α − 27.9 ± 3.5 32.8 ±2.9 + 10.1 ± 1.6 64 12.9 ± 2.3 61 IFN-γ −  87.9 ± 10.2 132.4 ± 11.6 + 9.9 ± 1.8 89 13.2 ± 2.4 90 TNF-α + IL-1β − 136.9 ± 15.8 182.6 ± 20.2 +22.8 ± 2.7 83 28.2 ± 4.3 85 TNF-α + IFN-γ − 145.9 ± 17.4 201.6 ± 22.5 +29.6 ± 4.2 79 36.9 ± 3.4 82 LPS + TNF-α − 168.2 ± 19.4 232.7 ± 20.6 +13.6 ± 2.6 92 19.7 ± 2.7 91 LPS + IFN-γ − 162.3 ± 15.2 218.5 ± 23.6 +25.2 ± 3.9 84 32.3 ± 4.6 83 LPS + IL-1β − 149.6 ± 11.2 204.3 ± 17.6 +28.4 ± 3.7 81 44.9 ± 6.1 78Nitrite accumulation in the supernatants and NOS activity in the cellswere measured as described. When different stimuli were used alone,their concentrations were: LPS, 1.0 μg/ml; TNF-α, 100 ng/ml; IL-1β, 200ng/ml; IFN-γ, 200 U/ml. When stimuli were used in different combinationstheir concentrations were: LPS, 0.2 μg/ml; TNF-α, 20 ng/ml; IL-1β, 25ng/ml; IFN-γ, 50 U/ml. Cells received NAC 2 h before the addition ofdifferent stimuli. Data are mean±S.D. of three different experiments.

NAC Inhibits LPS And/Or Cytokine-Mediated NO Production In RatAstrocytes And C6 Glial Cells A study was carried out to determine ifNAC inhibits LPS and/or cytokine-mediated NO production in ratastrocytes and glial cells. Both astrocytes and C6 glial cells arereported to express iNOS in the presence of different stimuli (Feinsteinet al., 1994a; Hu et al., 1995). Similar to previous reports (Feinsteinet al., 1994a; Feinstein et al., 1994b), incubation of C6 cells withLPS, TNF-α, IFN-γ or IL-1β alone did not stimulate nitrite productionwhereas addition of several combinations of either LPS or cytokinesinduced the production of NO (Table 6). In contrast to the induction ofNO production found in C6 glial cells, either LPS or cytokines aloneinduced the production of NO in cultured rat astrocytes (Table 6).However, similar to macrophages, addition of NAC at 20 mM concentration2 h prior to the addition of several cytokines blocked the induction ofNO production in both C6 glial cells and astrocytes (Table 6).

TABLE 6 Inhibition of nitrite production by NAC in stimulated astrocytesand C6 glial cells Astrocytes C6 glial cells NAC Nitrite Nitrite (20(nmol/ Inhibition (nmol/ Inhibition Stimuli mM) mg/24 h) (%) mg/24 h)(%) Control − 4.3 ± 1.2 3.1 ± 0.4 + — — — — LPS − 27.8 ± 5.2  3.5 ±0.4 + 5.6 ± 1.6 80 — — TNF-α − 23.4 ± 3.9  3.6 ± 0.2 + 10.5 ± 2.3  55 —— IL-1β − 30.1 ± 2.6  3.8 ± 0.5 + 7.8 ± 1.8 74 — — IFN-γ − 22.3 ± 4.2 5.3 ± 1.1 + 4.4 ± 1.2 80 — — LPS + TNF-α − 41.8 ± 2.8  33.9 ± 4.2  +15.2 ± 1.5  64 7.1 ± 1.1 79 LPS + IL-1β − 21.7 ± 2.9  22.8 ± 3.4  + 4.5± 2.8 79 5.8 ± 1.2 75 LPS + IFN-γ − 50.7 ± 6.8  32.1 ± 4.2  + 23.5 ±1.4  54 8.2 ± 1.4 74 TNF-α + IL-1β − 36.4 ± 4.5  20.7 ± 3.1  + 5.2 ± 0.786 4.2 ± 0.8 80 TNF-α + IFN-γ − 38.4 ± 2.6  30.9 ± 5.1  + 14.1 ± 2.04 638.4 ± 2.1 73Astrocytes and C6 glial cells were cultured for 24 h in serum-freeDMF-12 medium with the listed stimuli, and nitrite accumulation in thesupernatants was measured as described previously. Concentrations ofdifferent stimuli were the same as described in the legend of Table 4.Data are mean±S.D. of three different experiments.

Inhibition of LPS-mediated TNF-α production by NAC and PDTC inmacrophages LPS stimulates a variety of cell types including macrophagesto induce the production of TNF-α. To study whether antioxidants effectthe production of TNF-α, rat resident macrophages were cultured inserum-free RPMI 1640 medium. They were either treated with antioxidants(NAC and PDTC) 2 h before the addition of LPS or IFN-γ alone or incombination (Table 7). LPS alone at a concentration of 1.0 μg/ml inducedappreciable amounts of TNF-α production (20.3±3.6 ng/mg/24 h) and theaddition of IFN-γ augments the action of LPS (Table 7). However, IFN-γalone, was ineffective in inducing TNF-α from macrophages.Pre-incubation of cells with either NAC or PDTC almost completelyeliminated the induction of TNF-α production by LPS and IFN-γ. Theseresults indicate that similar to the induction of iNOS, the productionof TNF-α by LPS and IFN-γ involves oxygen radicals signal pathway.

TABLE 7 Effect of NAC and PDTC on TNF-α production in rat residentmacrophages stimulated with LPS and IFN-γ TNF-α Reagents (ng/24 h/mgprotein) Inhibition (%) Control 0.7 ± 0.1 NAC 0.6 ± 0.2 PDTC 0.7 ± 0.2LPS 20.3 ± 3.6  IFN-γ 1.3 ± 0.4 LPS + IFN-γ 31.6 ± 2.9  LPS + NAC 1.6 ±0.3 92 LPS + PDTC 0.9 ± 0.1 95 LPS + IFN-γ + NAC 2.3 ± 0.2 93 LPS +IFN-γ + PDTC 1.4 ± 0.3 96Resident macrophages cultured in serum-free RPMI 1640 medium received 20mM NAC or 100 μM PDTC 2 h before the addition of stimuli. When stimuliwere used separately their concentrations were: LPS, 1.0 μg/ml; IFN-γ,200 U/ml, and when they were used together, their concentrations were:LPS, 0.2 μg/ml; IFN-γ, 50 U/ml. Supernatants were collected 24 h afterthe addition of stimuli to measure TNF-α concentration using ELISA asdescribed.

EXAMPLE 3 Modulation of LPS-induced NO Production and Expression of iNOSin Rat Primary Astrocytes by Compounds that Modulate IntracellularLevels of cAMP

The activation of PKA correlates with the inhibition of LPS-induced iNOSexpression in rat primary astrocytes. Primary astrocytes in serum-freeDMEM/F-12 received 10 μM forskolin, 500 μM 8-Br-cAMP, 5 μM (S_(p))-cAMP,0.2 μM H-89, or 20 μM (R_(p))-cAMP 15 min before the addition of 1.0μg/ml LPS. Nitrite concentrations were measured in supernatants after 24h; and NOS activities were measured in cell homogenates as describedherein. Cell homogenates were electrophoresed, transferred onnitrocellulose membrane, and immunoblotted with antibodies against mousemacrophage iNOS. After 6 h of incubation, cells were taken out fromculture dishes directly by adding Ultraspec-II RNA reagent (BiotecxLaboratories Inc.) to isolate total RNA, and Northern blot analyses foriNOS mRNA were carried out as described in Examples 7 and 8. After 30min of incubation, PKA activities were measured in cells byphosphorylation of Kemptide in the presence or absence of the inhibitorpeptide PKI. Results were determined as the mean±S.D. of three differentstudies. Assay were conducted for control, LPS, LPS+forskolin,LPS+8-bromo-cAMP, LPS+(S_(p))-cAMP, LPS+H-89, and LPS+(R_(p))-cAMP.

The compounds forskolin, 8-bromo-cAMP, and (S_(p))-cAMP, known toincrease intracellular cAMP, inhibited the LPS-stimulated NO productionas nitrite, iNOS activity as conversion of arginine to citrulline,expression of iNOS protein and iNOS mRNA, and activated PKA activity.The inactive forskolin analogue, 1,9-dideoxyforskolin (10 μM), neitherinhibited the LPS induced iNOS activity nor stimulated the PKA activity(Table 8). Other PKA activators like β-adrenergic receptor agonist,isoproterenol (10 μM), and cAMP phosphodiesterase inhibitor,3-isobutyl-1-methylxanthine (1 mM), also inhibited LPS-stimulated NOproduction and iNOS activity (Table 8). On the other hand,LPS-stimulated NO production, iNOS activity, and expression of iNOSprotein and mRNA were increased by PKA inhibitors (H-89 and(R_(p))-cAMP). However, in the absence of LPS neither PKA activators norPKA inhibitors had any effect on the production of NO. This inhibitionof NO production by cAMP was not only confined to astrocytes, butforskolin was also found to inhibit LPS, and cytokine-induced NOproduction in rat C₆ glial cells C₆ glial cells incubated in serum-freeDMEM/F-12 received 10 μM forskolin 15 min before the addition of LPS andcytokines. Nitrite concentrations were measured in supernatants after 24h of incubation as described in Example 6. Concentrations of differentstimuli were included LPS, 0.5 μg/ml; TNF-α, 20 ng/ml; IL-1β, 50 ng/ml;and IFN-γ, 50 units/ml. Data were measured as the mean±S.D. of threedifferent studies. Assay were conducted for control, LPS, LPS+TNF-α,LPS+IFN-γ, LPS+IL-1β, LPS+TNF-α+forskolin, LPS+forskolin+IFN-γ, andLPS+forskolin+IL-1β.

The decrease in LPS-induced iNOS expression with the increase in cAMPlevel and the increase in LPS-induced iNOS expression with the decreasein cAMP level clearly delineate cAMP and cAMP-dependent protein kinaseas important regulators of iNOS biosynthesis in glial cells.

TABLE 8 Inhibition Of LPS-Induced Nitrite Accumulation In Rat PrimaryAstrocytes By Different cAMP Agonists Nitrite Inhibition Stimulinmol/mg/24 h % Control 3.2 ± 0.4 LPS 31.4 ± 3.6   0 LPS + forskolin 4.7± 1.2 85 LPS + dideoxyforskolin 31.2 ± 4.1  — LPS + isoproterenol 8.1 ±2.3 74 LPS + IBMX 6.8 ± 1.3 78 LPS + Rolipram 6.2 ± 1.2 80Primary astrocytes were cultured for 24 h in serum-free DMEM/F-12 withthe listed reagents; nitrite concentration in the supernatants was thenmeasured as described above. Concentration of reagents were: LPS, 1.0μg/ml; forskolin, 10 μM; 1,9-dideoxyforskolin, 10 μM; isoproterenol, 10μM; 1-isobutyl-1-methylxanthine (IBMX), mM; Rolipram, 10 μM. All thecAMP agonists were added to the cells 15 min prior to the addition ofLPS. Data are mean±S.D. of three different studies.

Dose Dependence of Forskolin Inhibition of the LPS Stimulation of iNOSAstrocytes were incubated with different concentrations of forskolin 15min before the addition of 1 μg/ml LPS, and after 24 h the iNOS activitywas measured as nitrite concentrations in the supernatant and conversionof arginine to citrulline in the cellular homogenates (FIG. 1). Thelevel of nitrite and iNOS activity were inhibited to a similar degree atall the concentrations of forskolin tested. The lowest does of forskolinfound to inhibit iNOS activity and NO production significantly (by 30%)was 0.1 μM. At 10 μM forskolin, NO production and iNOS activity wereinhibited by about 90%. Higher doses of forskolin (50-100 μM) did notresult in further significant inhibition of iNOS. This may be due to thefact that PKA was already completely activated in extracts of cellsincubated with 10 μM forskolin. The PKA activity increased with theincrease in forskolin concentration. The reciprocal relationship ofproduction of NO and iNOS activity with PKA activity supports theconclusion that PKA plays a pivotal role in the regulation of iNOSexpression in astrocytes.

Modulation of LPS- and/or Cytokine-mediated iNOS Expression by CompoundsModulating Intracellular Levels of cAMP in Rat Primary AstrocytesPrimary astrocytes were stimulated with TNF-α, IL-1β, and IFN-γ alone orin different combinations for 24 h and iNOS was measured. TNF-α, IL-1β,and IFN-γ individually were able to induce iNOS activity, protein, andmRNA, however, when tested in combinations between them or with LPS, themagnitude of induction was significantly higher. Cells incubated inserum-free DMEM/F-12 received 10 μM forskolin or 0.2 μM H-89 15 minbefore the addition of the different cytokines (TNF-α, 100 ng/ml; IL-1β,200 ng/ml; IFN-γ, 200 units/ml). Assay were conducted for control,TNF-α, IL-1β, IFN-γ, TNF-α+H-89, IL-1β+H-89, IFN-γ+H-89,TNF-α+forskolin, IL-1β+forskolin, IFN-γ+forskolin. Activities for iNOSwere measured in cell homogenates after 24 h as described. Results areexpressed as means±S.D. of three different studies. Cell homogenateswere immunoblotted with antibodies against mouse macrophage iNOS asdescribed. After 6 h of incubation, cells were taken out from culturedishes directly by adding Ultraspec-II RNA reagent (Biotecx LaboratoriesInc.) to isolate total RNA and northern blot analyses for iNOS mRNA werecarried out as described. Forskolin, the activator of PKA, completelyinhibited the cytokine-induced expression of iNOS, whereas H-89, aspecific inhibitor of PKA, stimulated the cytokine-induced expression ofiNOS.

Similarly, the induction of iNOS by several combinations of cytokinesand LPS were also inhibited by forskolin in rat primary astrocytes,indicating that augmentation of the cellular levels of cAMP and theactivation of cAMP-dependent protein kinase represents a generalcounter-regulatory mechanism for down-regulation of iNOS expression inastrocytes. Cells in this study were incubated in serum free DMEM/F-12received 10 μm forskolin (FOR) for 15 min before the addition ofdifferent combinations of LPS and cytokines. After 24 h of incubation,cell homogenates were analyzed for: iNOS activity, and iNOS protein byimmunoblotting. After 6 h of incubation, cells were taken out andnorthern blot analyses for iNOS mRNA were carried out as described.Concentrations of different stimuli were: LPS, 0.5 μg/ml; TNF-α, 20ng/ml; IL-1β, 50 ng/ml; IFN-γ, 50 units/ml. Assay were conducted forcontrol, TNF-α+IFN-γ, TNF-α+IL-1β, LPS+IL-1β, LPS+TNF-α, LPS+IFN-γ,TNF-α+IFN-γ+forskolin, TNF-α+IL-1β+forskolin, LPS+IL-1β+forskolin, andLPS+IFN-γ+forskolin.

EXAMPLE 4 Therapy for X-Adrenoleukodystrophy: Normalization of Very LongChain Fatty Acids and Inhibition of Induction of Cytokines by cAMP

Materials and Methods

Reagents DMEM and bovine calf serum were from GIBCO. Forskolin,1,9-dideoxyforskolin, 8-Br cAMP, S(p)-cAMP, H-89, rp-cAMP and rolipramwere obtained from Biomol, USA. C_(18:0)-CoA, NADPH and N-ethylmaleimidewere from Sigma (USA). [2-¹⁴C]Malonyl-CoA and K¹⁴CN (52 mCi/mmol) werepurchased from DuPont-New England Nuclear. [1-¹⁴C]Lignoceric acid wassynthesized by treatment of n-tricosanoyl bromide with K¹⁴CN asdescribed previously (Hoshi and Kishimoto, 1973).

Enzyme assay for β-oxidation of lignoceric acid The enzyme activity of[1-¹⁴C]lignoceric acid β-oxidation to acetate was measured in intactcells suspended in Hank's Buffered Salt Solution (HBSS). Briefly, thereaction mixture in 0.25 ml of HBSS contained 50-60 μg of protein and 6μM [1-¹⁴C]lignoceric acid. Fatty acids were solubilized withα-cyclodextrin and β-oxidation of [1-¹⁴C]lignoceric acid was carried outas described previously (Singh et al., 1984; Hashmi et al., 1986;Lageweg et al., 1991; Lazo et al., 1988). The reaction was stopped after1 h with 0.625 ml of 1 M KOH in methanol, and the denatured protein wasremoved by centrifugation. The supernatant was incubated at 60° C. for 1h, neutralized with 0.125 ml of 6 N HCl, and partitioned with chloroformand methanol. Radioactivity in the upper phase is an index of[1-¹⁴C]lignoceric acid oxidized to acetate.

Transport of lignoceric acid into cultured skin fibroblasts Cells wereincubated for 15 min at 37° C. under isotonic conditions in HBSS with[1-¹⁴C]lignoceric acid (6 μM) solubilized with α-cyclodextrin asdescribed earlier (Singh et al., 1984; Hashmi et al., 1986; Lageweg etal., 1991). Then cells were separated from the incubation medium bycentrifugation through an organic layer of brominated hydrocarbons(Cornell, 1980). This was performed in micro tubes (1.5 ml) containing50 μl of 0.25 M sucrose in HBSS (as cushion), an organic layer (400 μl)consisting of a mixture of bromododecane and bromodecane (7:4, v/v), andan upper layer (500 μl) of cells in HBSS.

Protein kinase A assay Cell extracts were assayed for PKA activity asdescribed (Graves et al., 1993) and herein by measuring thephosphorylation of kemptide (0.17 mM) in the presence or absence of PKIpeptide (15 μM). PKA activity was calculated as the amount of kemptidephosphorylated in the absence of PKI peptide minus that phosphorylatedin the presence of PKI peptide.

Enzyme assay for fatty acid elongation The fatty acid elongationactivity was assayed by the method of Tsuji et al. (Tsuji et al., 1984).Briefly, the assay mixture contained 100 mM potassium phosphate (pH7.2), 0.5 mM NADPH, 0.05 mM [2-¹⁴C]malonyl-CoA, 1 mM N-ethyl maleimideand 50-60 μg of protein in a total volume of 0.25 ml. The concentrationsof C_(18:0)-CoA was 1 mM. The reaction was started at 37° C. by theaddition of total homogenate and stopped by the addition of 1.25 ml of10% (w/v) KOH after 30 min incubation. After saponification at 100° C.for 30 min, the solutions were acidified with 1 ml of 4N HCl and fattyacids were extracted with 2.5 ml of n-pentane three times. Theradioactivities incorporated into fatty acids were measured with aliquid scintillation counter.

Measurement of VLCFA in Fibroblasts Fatty acid methyl ester (FAME) wasprepared as described previously by Lepage and Roy (1986) withmodifications. Fibroblast cells, suspended in HBSS, were disrupted bysonication to form a homogeneous solution. An aliquot (200 μl) of thissolution was transferred to a glass tube and 5 g heptacosanoic (27:0)acid was added as internal standard and lipids were extracted by Folchpartition. Fatty acids were transesterified with acetyl chloride (200μl) in the presence of methanol and benzene (4:1) for 2 h at 100° C. Thesolution was cooled down to room temperature followed by addition of 5ml 6% potassium carbonate solution at ice-cooled temperature. Isolationand purification of FAME were carried out as detailed by Dacremont etal. (1995). Purified FAME, suspended in chloroform, were analyzed by gaschromatograph GC-15A attached with chromatopac C-R3A integrator fromSchimadzu Corporation.

Preparation of post-nuclear membrane and western blot analysis TheMembranes were prepared as described previously (Contreras et al.,1996). Briefly, the post-nuclear fraction was diluted with an ice-coldsolution of 0.1 M sodium carbonate, 30 mM iodoacetamide, pH 11.5. After30 min of incubation at 4° C., the membranes were sedimented byultracentrifugation. The sedimented membranes were electrophoresed in7.5% sodium dodecylsulfate-polyacrylamide gel, transferred to PVDFmembranes and immunoblotted with antibodies against ALDP as described(Contreras et al., 1996).

RNA isolation and Northern blot analysis Cultured skin fibroblasts weretaken out from culture flasks directly by adding Ultraspec-II RNAreagent (Biotecx Laboratories Inc.) and total RNA was isolated accordingto the manufacturer's protocol. Twenty micrograms of RNA from eachsample were electrophoretically resolved on 1.2% denaturingformaldehyde-agarose gel, transferred to nylon membrane, andcross-linked using UV Stratalinker (Stratagene, USA). Full length ALDPcDNA was obtained from Dr. Patrick Aubourg, INSERM, HospitalSaint-Vincent-de-Paul, Paris, France. ³²P-labeled cDNA probes wereprepared according to the instructions provided with Ready-To-Go DNAlabeling kit (Pharmacia Biotech). Northern blot analysis was performedessentially as described for Express Hyb Hybridization solution(Clontech) at 68° C. Actin cDNA probe was used as standard for comparinghybridization signals.

Isolation of rat primary astrocytes and microglia Astrocytes wereprepared from rat cerebral tissue as described (McCarthy and De Vellis,1980) and herein. Microglial cells were isolated from mixed glialcultures according to the procedure of Guilian and Baker (1986). For theinduction of cytokine production, cells were stimulated with LPS inserum-free condition.

Determination of TNF-α and IL-1β in culture supernatants Cells werestimulated with LPS in serum-free media for 24 h in the presence orabsence of forskolin or rolipram, and concentrations of TNF-α and IL-1βwere measured in culture supernatants by using high-sensitivityenzyme-linked immunosorbent assay (R&D Systems, USA) according to themanufacturer's instructions.

Results

Compounds that modulate the intracellular cAMP also modulate theβ-oxidation of lignoceric acid and fatty acid chain elongation in X-ALDfibroblasts: First, the effect of cAMP derivatives on lignoceric acidβ-oxidation in control human fibroblasts was examined. Cultured skinfibroblasts were treated with different activators and inhibitors ofprotein kinase A (PKA) and tested for β-oxidation of lignoceric acid. Itis apparent from Table 9 that compounds known to increase cAMP(forskolin and 8-Br-cAMP) stimulated lignoceric acid β-oxidation whereascompounds known to decrease cAMP (H-89 and myristoylated PKI) inhibitedlignoceric acid β-oxidation in control skin fibroblasts. The inactiveanalogue of forskolin, 1,9-dideoxyforskolin, was ineffective instimulating β-oxidation (Table 9). These results indicate that PKA has apositive modulatory role on lignoceric acid β-oxidation. Since theβ-oxidation of lignoceric acid is impaired in X-ALD patients, theinventor studied the effect of different activators and inhibitors ofPKA on lignoceric acid β-oxidation in cultured skin fibroblasts ofX-ALD. FIG. 2 shows that the compounds (forskolin, 8-bromo cAMP androlipram) known to increase intracellular cAMP stimulated lignocericacid β-oxidation (FIG. 2A) and activated the PKA activity (FIG. 2C). Onthe other hand, β-oxidation of lignoceric acid was inhibited by PKAinhibitors (H-89 and myristoylated PKI). A combination of forskolin(activator of PKA) and H-89 or myristoylated PKI (inhibitors of PKA) hadrelatively little effect on the activation of PKA as well as on theβ-oxidation of lignoceric acid. These observations indicate thatβ-oxidation of lignoceric acid is modulated by cAMP and PKA.

However, in contrast to the effects on β-oxidation of lignoceric acid,activators of PKA inhibited the fatty acid chain elongation andinhibitors of PKA stimulated this activity in X-ALD fibroblasts (FIG.2B). The increase in β-oxidation of lignoceric acid by forskolin andit's inhibition by H-89 were dose-dependent. Cells in this experimentwere incubated in serum-containing DMEM with different concentrations offorskolin (0-10 μM) or H-89 (0-4 μM) for 72 h. After every 24 h, mediawas replaced with the addition of fresh reagents. β-oxidation oflignoceric acid (pmol/h/mg protein) was measured in cell-suspension asdescribed in the methods section.

To understand the mechanism of cAMP-mediated stimulation of lignocericacid β-oxidation, fibroblasts of X-ALD were treated with cAMP analogs,and the transport of lignoceric acid into intact cells and β-oxidationof lignoceric acid in cell homogenates of X-ALD were measured. Similarto the modulation of lignoceric acid β-oxidation, activators of PKA alsostimulated the transport of lignoceric acid into ALD cells by more thantwo fold whereas inhibitors of PKA inhibited the transport of lignocericacid by 40 to 50 percent. Stimulation of lignoceric acid β-oxidation incell homogenates of ALD fibroblasts as well as in cell suspension (FIG.2A) indicates that increase in β-oxidation is not due to anintracellular increase of substrate concentration but by stimulation ofenzyme system for oxidation of lignoceric acid. In the cell, fatty acidsare oxidized by mitochondrial and peroxisomal β-oxidation enzyme.Etomoxir, an inhibitor of mitochondrial β-oxidation of fatty acids(Mannaerts et al., 1979), had no effect on cAMP-mediated stimulation oflignoceric acid β-oxidation indicating that the observed stimulation oflignoceric acid was a peroxisomal function. The increase in β-oxidationand transport of lignoceric acid but the decrease in fatty acid chainelongation with the increase in cAMP level and PKA activity, and thedecrease in β-oxidation and transport of lignoceric acid but theincrease in fatty acid chain elongation with the decrease in cAMP leveland PKA activity clearly delineate cAMP and cAMP-dependent proteinkinase A as important regulators of the metabolism of VLCFA.

TABLE 9 Effects of different agonists and antagonists of PKA onβ-oxidation of lignoceric acid in control human fibroblasts Lignocericacid β-oxidation Treatments (pmol/h/mg protein) Control 565.2 ± 48.3Forskolin 885.3 ± 62.1 1,9 dideoxy forskolin 571.4 ± 39.6 8-Br-cAMP872.0 ± 53.7 H-89 405.6 ± 44.1 Myristoylated PKI 432.3 ± 46.5Cells were treated for 72 h serum-containing DMEM with the listedreagents; β-oxidation of lignoceric acid was measured as described under“Material and Methods”. Media was replaced after every 24 h with theaddition of fresh reagents. Concentrations of reagents were: forskolin,4 and μM; 1,9 dideoxy forskolin, 4 μM; 8-Br-cAMP, 50 μM; H-89, 1 μM;myristoylated PKI, 0.2 μM. Data are mean±S.D. of three differentexperiments.

Modulation of cellular content of VLCFA in X-ALD and AMN fibroblasts bycompounds modulating intracellular levels of cAMP Since cAMP derivativesincrease β-oxidation of lignoceric acid and decrease fatty acid chainelongation, the effect of cAMP derivatives on the level of VLCFA inX-ALD fibroblasts was examined. Treatment of X-ALD fibroblasts with 4 μMof forskolin for different time periods (days) resulted in atime-dependent increase in oxidation of lignoceric acid and atime-dependent decrease C_(22:0) in the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0) as shown in FIG. 3A-3C. Within 12 to 15 days oftreatment, the ratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) inX-ALD fibroblasts decreased to the normal level. This decrease in theratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) was also associatedwith the decrease in the absolute amounts of C_(24:0) and C_(26:0)whereas no significant change was observed in the levels of C_(22:0)(behenoic acid). To decipher the possible mechanism of this dramaticdecrease of VLCFA, X-ALD fibroblasts were treated with differentactivators of PKA (forskolin, 8-Br-cAMP and rolipram) for 15 days andanalyzed the level of VLCFA. The treatment of X-ALD cultured skinfibroblasts with compounds known to increase intracellular cAMP loweredthe ratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) to the normallevel. Cells were incubated in serum-containing DMEM for 15 days withcontrol, forskolin, 8-Br-cAMP, rollpram, forskolin+H-89, H-89, andIFN-β, and the ratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) weremeasured as described in Example 4. Concentrations of reagents were:forskolin, 4 μM; 8-Br-cAMP, 50 μM; rolipram, 10 μM; H-89, 1 μM;myristoylated PKI, 0.2 μM; IFN-β, 50 U/ml. Data are mean±S.D. of threedifferent experiments. The inactive forskolin analogue,1,9-dideoxyforskolin, had no effect on the ratios of C_(26:0)/C_(22:0)and C_(24:0)/C_(22:0). However, compared to X-ALD fibroblasts, forskolinmarginally lowered the ratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0)in control skin fibroblasts. A two weeks treatment with forskolinlowered the ratio of C_(26:0)/C_(22:0) from 0.04 to 0.029 and the ratioof C_(24:0)/C_(22:0) from 1.23 to 1.12. Consistent with the effect ofH-89 and myristoylated PKI on the β-oxidation of lignoceric acid, thesetwo compounds blocked the observed effect of forskolin on the level ofVLCFA when added along with forskolin indicating that cAMP analogs lowerthe level of VLCFA in X-ALD fibroblasts via activation of PKA. On theother hand, interferon-β, which has been indicated as a possible therapyfor X-ALD based on favorable effects found in multiple sclerosis (Moser,1995), was ineffective in lowering the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0) in skin fibroblasts of X-ALD.

Normalization of the levels of VLCFA by forskolin or rolipram indifferent X-ALD cells with or without deletion of the X-ALD geneAlthough the precise function of ALDP, an X-ALD gene product, in themetabolism of VLCFA is not known at the present time, accumulation ofVLCFA in X-ALD cells with loss or mutations of ALDP and theirnormalization following transfection of cDNA for ALDP indicate a role ofALDP in the metabolism of VLCFA (Cartier et al., 1995). Therefore, theinventor examined whether decrease in VLCFA in X-ALD fibroblasts byactivators of PKA is mediated through the involvement of the ALD gene.ALDS1, ALDS5 and ALDS6 are X-ALD skin fibroblasts with deletion of theX-ALD gene, whereas ALDS2, ALDS3 and ALDS4 are X-ALD skin fibroblastswith mutation of the X-ALD gene. These cell lines were incubated inserum-containing DMEM with 4 μM forskolin for 15 days or control medium,and the ratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0), β-oxidationof lignoceric acid and elongation of fatty acids were measured asdescribed in Example 4. X-ALD cells with mutation or deletion of the ALDgene were treated with forskolin for two weeks and tested for the levelsof ALDP protein and it's mRNA, levels of VLCFA. Results were measured asthe mean±S.D. of three different experiments.

It was apparent that treatment of X-ALD fibroblasts with forskolin fortwo weeks had no effect on the steady state levels of ALDP and its mRNAin X-ALD cells. However, forskolin normalized the level of VLCFA inX-ALD fibroblasts by decreasing the rate of fatty acid chain elongationand increasing the rate of β-oxidation of lignoceric acid despite thestatus of mRNA and protein of ALDP. Treatment of X-ALD fibroblasts withrolipram for two weeks also increased the oxidation of lignoceric acidbetween 50 to 65 percent and normalized the levels of VLCFA in thesecell lines indicating that rolipram, an inhibitor of cAMPphosphodiesterase, has same effect on the metabolism of VLCFA in X-ALDcells with nonfunctional ALDP due to a mutation or with absence of ALDPdue to a deletion of the X-ALD gene.

Forskolin and rolipram inhibit the induction of cytokine production inrat primary astrocytes and microglia Since both astrocytes andmicroglia, reactive glial cells, in the demyelinating lesions of ALDbrain, are reported to express TNF-α and IL-1β (Powers et al., 1992;McGuinness et al., 1995), the effect of cAMP derivatives on theinduction of cytokine production in astrocytes and microglia wasstudied. Primary astrocytes in serum-free DMEM/F-12 were treated withdifferent activators and inhibitors of PKA 15 min before the addition of1 μm of lipopolysaccharide (LPS). FIG. 4 shows that the compounds(forskolin, 8-bromo-AMP, and rolipram) known to increase intracellularcAMP inhibited the LPS-stimulated production of TNF-α (FIG. 4A) andIL-1β (FIG. 4B), and activated PKA activity (FIG. 4C). On the otherhand, LPS-stimulated production of TNF-α and IL-1β were increased byinhibitors of PKA (H-89 and myristoylated PKI). The reciprocalrelationship of induction of TNF-α and IL-1β with PKA activity supportsthe conclusion that PKA plays a pivotal role in the regulation ofproinflammatory cytokines in astrocytes. Similar to astrocytes,forskolin or rolipram also inhibited the LPS-induced production of TNF-αand IL-1β, and H-89 stimulated the production of these proinflammatorycytokines in rat primary microglia (Table-10).

TABLE 10 Inhibition of LPS-induced production of TNF-α and IL-1β in ratprimary microglia by forskolin and rolipram Production of Treatmentscytokines LPS only LPS + Forskolin LPS + Rolipram TNF-α 14.1 ± 2.1 0.9 ±0.1 1.2 ± 0.09 IL-1β 20.8 ± 2.8 1.9 ± 0.2 2.3 ± 0.3 Cells preincubated with 10 μM forskolin or 20 μM of rolipram for 15 minin serum-free condition was stimulated with 1.0 μg/ml of LPS. After 24 hof incubation, concentrations of TNF-α and IL-β were measured insupernatants as described in the methods section. TNF-α and IL-β areexpressed as ng/24 h/mg protein. Data are expressed as the mean±S.D. ofthree different experiments.

EXAMPLE 5 Lovastatin and Sodium Phenylacetate Normalize the Level ofVery Long Chain Fatty Acids in Skin Fibroblasts ofX-Adrenoleukodystrophy

Materials and Methods

Reagents DMEM, bovine calf serum and Hank's Buffered Salt Solution(HBSS) were from GIBCO. [1-¹⁴C]Lignoceric acid was synthesized bytreatment of n-tricosanoyl bromide with K¹⁴CN as described previously.

Enzyme assay for β-oxidation of lignoceric acid The enzyme activity of[1-¹⁴C]lignoceric acid β-oxidation to acetate was measured in intactcells suspended in HBSS. Briefly, the reaction mixture in 0.25 ml ofHBSS contained 50-60 μg of protein and 6 μM [1-¹⁴C]lignoceric acid.Fatty acids were solubilized with α-cyclodextrin and β-oxidation of[1-¹⁴C]lignoceric acid was carried out as described previously (Singh etal., 1984; Lazo et al., 1988).

Measurement of VLCFA in Fibroblasts Fatty acid methyl ester (FAME) wasprepared as described previously (Lepage and Roy, 1986) withmodifications. Fibroblast cells, suspended in HBSS, were disrupted bysonication to form a homogeneous solution. An aliquot (200 μl) of thissolution was transferred to a glass tube and 5 μg heptacosanoic (27:0)acid was added as internal standard and lipids were extracted by Folchpartition. Fatty acids were transesterified with acetyl chloride (200μl) in the presence of methanol and benzene (4:1) for 2 h at 100° C. Thesolution was cooled down to room temperature followed by addition of 5ml 6% potassium carbonate solution at ice-cooled temperature. Isolationand purification of FAME were carried out as detailed by Dacremont etal. (1995). Purified FAME, suspended in chloroform, were analyzed by gaschromatograph GC-15A attached with chromatopac C-R3A integrator fromShimadzu Corporation.

Preparation of post-nuclear membrane and western blot analysis TheMembranes were prepared as described previously (Contreras et al.,1996). Briefly, the post-nuclear fraction was diluted with an ice-coldsolution of 0.1 M sodium carbonate, 30 mM iodoacetamide, pH 11.5. After30 min of incubation at 4° C., the membranes were sedimented byultracentrifugation. The sedimented membranes were electrophoresed in7.5% sodium dodecylsulfate-polyacrylamide gel, transferred to PVDFmembranes and immunoblotted with antibodies against ALDP as described(Contreras et al., 1996).

RNA isolation and Northern blot analysis Cultured skin fibroblasts weretaken out from culture flasks directly by adding Ultraspec-II RNAreagent (Biotecx Laboratories Inc.) and total RNA was isolated accordingto the manufacturer's protocol. Twenty micrograms of RNA from eachsample were electrophoretically resolved on 1.2% denaturingformaldehyde-agarose gel, transferred to nylon membrane, andcross-linked using UV Stratalinker (Stratagene, USA). Full length ALDPcDNA was obtained from Dr. Patrick Aubourg, INSERM, HospitalSaint-Vincent-de-Paul, Paris, France. ³²P-labeled cDNA, probes wereprepared according to the instructions provided with Ready-To-Go DNAlabeling kit (Pharmacia Biotech). Northern blot analysis was performedessentially as described for Express Hyb Hybridization solution(Clontech) at 68° C. GAPDH cDNA probe was used as standard for comparinghybridization signals.

Results

Inhibitors of mevalonate pathway stimulate the β-oxidation of lignocericacid in X-ALD fibroblasts First, the effect of mevalonate inhibitors(lovastatin, mevastatin and NaPA) on the β-oxidation of lignoceric acidin control human fibroblasts was examined. It is apparent from Table 11that lovastatin, mevastatin and NaPA stimulated the β-oxidation oflignoceric acid in control human fibroblasts. Since the β-oxidation oflignoceric acid is impaired in X-ALD patients, the effect of thesecompounds on lignoceric acid β-oxidation was studied in cultured skinfibroblasts of X-ALD.

TABLE 11 Lovastatin and NaPA stimulate the β-oxidation of lignocericacid in control human skin fibroblasts Lignoceric acid β-oxidationTreatments (pmol/h/mg protein) Control 570.2 ± 52.3 Lovastatin (5 μM) 945.7 ± 105.6 Mevastatin (5 μM) 889.6 ± 78.4 NaPA (5 mM) 826.2 ± 87.2Cells were treated for 72 h in serum-containing DMEM with the listedreagents; β-oxidation of lignoceric acid was measured as described inthe methods section. Media was replaced after every 24 h with theaddition of fresh reagents. Data are mean±S.D. of three differentstudies.

Similar to control fibroblasts, these compounds also stimulatedlignoceric acid β-oxidation in X-ALD skin fibroblasts. Cells wereincubated in serum-containing DMEM with different concentrations oflovastatin (0-10 μM) or NaPA (0-5 mM). After every 24 h, media wasreplaced with the addition of fresh reagents. Lignoceric acidβ-oxidation was measured (pmol/h/mg protein) after 72 h incell-suspension as described in the methods section. Values weredetermined as the mean±S.D. of three different studies. Both lovastatinand NaPA dose-dependently stimulated lignoceric acid β-oxidation inX-ALD fibroblasts. The highest dose of lovastatin found to stimulatelignoceric acid β-oxidation (by 70%) was 5 μM whereas the highest doseof NaPA found to stimulate lignoceric acid β-oxidation (by 40%) was 5mM. However, greater degree of stimulation (more than two fold) wasobserved by a combination of lovastatin and NaPA even at a dose lowerthan the one used individually. Higher doses of lovastatin (10-20 μM) orNaPA (10-20 mM) were cytotoxic to the X-ALD fibroblasts and did notresult in further significant stimulation. In the cell, fatty acids areoxidized by mitochondrial and peroxisomal β-oxidation enzyme. Etomoxir,an inhibitor of mitochondrial β-oxidation of fatty acids (Mannaerts etal., 1979), had no effect on lovastatin- or NaPA-mediated stimulation oflignoceric acid β-oxidation indicating that the observed stimulation oflignoceric acid β-oxidation was a peroxisomal function.

Modulation of cellular content of VLCFA in X-ALD fibroblasts bylovastatin and NaPA Since mevalonate inhibitors increased β-oxidation oflignoceric acid in control as well as X-ALD fibroblasts, the inventorexamined the effect of these compounds on the level of VLCFA in X-ALDfibroblasts. Treatment of X-ALD cultured skin fibroblasts with 5 μM oflovastatin for different time periods (days) resulted in atime-dependent decrease in the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0). Cells were incubated in serum-containing DMEM with 5μM lovastatin, 5 mM NaPA or the combination of 4 μM lovastatin and 2 mMNaPA for 0-15 days, and the ratios of C_(26:0)/C_(22:0) (24A) andC_(24:0)/C_(22:0) (24B) were measured every 3 days as described in themethods section. Values are mean of two different studies. Within 12 to15 days of treatment, the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0) in X-ALD fibroblasts decreased to the normal level.Similar to lovastatin, NaPA also lowered the ratios of C_(26:0)/C_(22:0)and C_(24:0)/C_(22:0) in X-ALD fibroblasts almost to the normal levelafter 15 days of treatment. However, consistent with the higher degreeof stimulation of lignoceric acid β-oxidation by a combination oflovastatin and NaPA, the same combination lowered the ratios ofC_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) to the normal level within 7days. This decrease in the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0) was also associated with the decrease in the absoluteamounts of C_(24:0) and C_(26:0) whereas no significant change wasobserved in the levels of C_(22:0) (behenoic acid).

Normalization of the levels of VLCFA by lovastatin or NaPA in differentX-ALD cells with or without deletion of the X-ALD gene Although theprecise function of ALDP, X-ALD gene product, in the metabolism of VLCFAis not known at the present time, however, accumulation of VLCFA inX-ALD cells with loss or mutations of ALDP and their normalizationfollowing transfection of cDNA for ALDP indicate a role of ALDP in themetabolism of VLCFA (Cartier et al., 1995). Therefore, the inventorexamined whether lovastatin or NaPA were able to lower the level ofVLCFA in different X-ALD fibroblasts with mutation or deletion of theX-ALD gene. The status of ALDP mRNA or protein and the rate ofβ-oxidation of lignoceric acid (Table 12) in different X-ALD fibroblastsindicates that ALDS2, ALDS3 and ALDS4 are X-ALD skin fibroblasts withmutation of the X-ALD gene, whereas ALDS5 and ALDS6 are X-ALD skinfibroblasts with deletion of the X-ALD gene. It is apparent from Table 3that treatment of X-ALD fibroblasts with lovastatin or NaPA or thecombination of these two stimulated the β-oxidation of lignoceric acid(55-80%) and normalized the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0) indicating that these drugs are capable of loweringthe level of VLCFA in X-ALD fibroblasts to the normal level,irrespective of mutation or deletion of the X-ALD gene, the candidategene for X-ALD.

TABLE 12 Effect of lovastatin-and NaPA on (A) β-oxidation of lignocericacid and (B) the ratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) incultured skin fibroblasts of X-ALD A Lignoceric acid β-oxidation(pmol/h/mg protein) Control Lovastatin NaPA Lovastatin + NaPA ALDS2142.7 ± 15.7 223.5 ± 24.1 202.5 ± 17.4 274.6 ± 30.5 ALDS5 154.2 ± 14.2248.2 ± 26.2 211.5 ± 22.6 296.2 ± 25.6 ALDS6 132.4 ± 15.9 218.3 ± 19.8189.7 ± 21.2 250.1 ± 28.3 ALDS3 122.3 ± 11.7 201.3 ± 22.3 183.2 ± 17.3248.6 ± 29.6 ALDS4 118.5 ± 12.6 192.8 ± 20.5 178.9 ± 18.3 238.7 ± 21.1 BC_(26:0)/C_(22:0) C_(24:0)/C_(22:0) Cell Lines Control LovastatinLovastatin + NaPA Control Lovastatin Lovastatin + NaPA ALDS2 0.17 ±0.022 0.049 ± 0.01 0.04 ± 0.008 1.84 ± 0.25 1.25 ± 0.15 1.14 ± 0.15ALDS5 0.18 ± 0.025  0.055 ± 0.008 0.04 ± 0.007 1.94 ± 0.29 1.28 ± 0.211.18 ± 0.12 ALDS6 0.22 ± 0.034 0.058 ± 0.01 0.045 ± 0.008  2.01 ± 0.3 1.31 ± 0.18 1.21 ± 0.14 ALDS3 0.16 ± 0.024 0.045 ± 0.06 0.03 ± 0.0051.88 ± 0.21 1.26 ± 0.16 1.19 ± 0.25 ALDS4 0.19 ± 0.028 0.052 ± 0.070.036 ± 0.006  1.96 ± 0.23 1.29 ± 0.02 1.22 ± 0.15Cells were incubated in serum-containing DMEM with 5 μM lovastatin, 5 mMNaPA or the combination of 4 μM forskolin and 2 mM NaPA for 15 days, andthe β-oxidation of lignoceric acid (A) and the ratios ofC_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) (B) were measured as describedin the methods section. Results are mean±S.D. of three differentstudies. ALDS2, ALDS3 and ALDS4 are X-ALD skin fibroblasts with mutationof the X-ALD gene, whereas ALDS5 and ALDS6 are X-ALD skin fibroblastswith deletion of the X-ALD gene.

The results of the preceding Examples 1-4 that lovastatin and NaPAinhibit the induction of nitric oxide synthase and proinflammatorycytokines (TNF-α, IL-1β and IL-6) in rat primary astrocytes, microgliaand macrophages indicates that these drugs, alone or in combination,represent a novel approach for therapeutics directed against cytokine-and NO-mediated brain disorders, particularly in demyelinatingconditions. Lovastatin and NaPA have already been approved formedication/drug trials on human diseases. Therefore, normalization ofVLCFA by lovastatin and NaPA in X-ALD fibroblasts indicates that thesedrugs may be used to lower the level of VLCFA and ameliorate themyelinolytic inflammation in X-ALD patients.

EXAMPLE 6 Inhibitors of Phosphatase 1 and 2A Differentially RegulateExpression of iNOS

Materials and Methods

Reagents Recombinant rat IFN-γ, DMEM/F-12 medium, fetal bovine serum,Hanks' balanced salt solution (HBSS) and NF-kB DNA binding proteindetection kit were from GIBCO. Human IL1-β was from Genzyme. Mouserecombinant TNF-α was obtained from Boehringer Mannheim, Germany. LPS(Escherichia coli) was from Sigma. N^(G)-methyl-L-arginine (L-NMA),okadaic acid, calyculin A, cantharidin and antibodies against mousemacrophage iNOS were obtained from Calbiochem, USA. Deltamethrin andfenvalerate were obtained from Biomol, USA. [γ-³²P]ATP (3000 Ci/mmol)were from Amersham, USA.

Induction of NO production in astrocytes and C₆ glial cells Astrocyteswere prepared from rat cerebral tissue as described by McCarthy andDeVellis (McCarthy and DeVellis, 1980). Cells were maintained inDMEM/F-12 medium containing 10% fetal bovine serum (FBS).

After 10 days of culture astrocytes were separated from microglia andoligodendrocytes by shaking for 24 h in an orbital shaker at 240 rpm.The shaking was repeated two more times after a gap of one or two daystime before subculturing to ensure the complete removal of all theoligodendrocytes and microglia. Cells were trypsinized, subcultured andstimulated with LPS or different cytokines in serum-free DMEM/F-12. C₆glial cells obtained from ATCC was also maintained and induced withdifferent stimuli as above.

Isolation of rat macrophages and induction of NO production Residentmacrophages were obtained from rat by peritoneal lavage with sterileRPMI 1640 medium containing 1% fetal bovine serum and 100 μg/mlgentamicin as described herein. Cells were washed three times with RPMI1640 at 4° C. All cell cultures were maintained at 37° C. in ahumidified incubator containing 5% CO₂ in air. Macrophages at aconcentration of 2×10⁶/ml in RPMI 1640 medium containing L-glutamine andgentamicin were added in volumes of 800 μl to a 35 mm plate. After 1 h,nonadherent cells were removed by washing and 800 μl of serum-free RPMI1640 medium with various stimuli were added to the adherent cells. Afterincubation in 5% CO₂ in air at 37° C., culture supernatants weretransferred to measure NO production.

Assay for NO synthesis Synthesis of NO was determined by assay ofculture supernatants for nitrite, a stable reaction product of NO withmolecular oxygen. Briefly, 400 μl of culture supernatant was allowed toreact with 200 μl of Griess reagent and incubated at room temperaturefor 15 min. The optical density of the assay samples was measuredspectrophotometrically at 570 nm. Fresh culture media served as theblank in all studies. Nitrite concentrations were calculated from astandard curve derived from the reaction of NaNO₂ in the assay.

In vitro PP1/2A assay The extraction and assay for PP1/2A were performedas described (Begum and Ragolia, 1996). Control and treated cells werescraped off the dishes with 0.3 ml of phosphatase extraction buffercontaining 20 mM imidazole-HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0, with 10μg/ml each of aprotinin, leupeptin, antipain, soybean trypsis inhibitor,1 mM benzamide, and 1 mM PMSF. The cells were sonicated for 10 s andcentrifuged at 2000×g for 5 min, and the supernatants were used for theassay of phosphatase activities using the protein phosphatase assay kit(Life Technologies, Inc.) according to the manufacturer's protocol.

Immunoblot analysis for iNOS Following 24 h incubation in the presenceor absence of different stimuli, cells were scraped off, washed withHank's buffer, and homogenized in 50 mM Tris-HCl (pH 7.4) containingprotease inhibitors. After electrophoresis the proteins were transferredonto a nitrocellulose membrane, and the iNOS band was visualized byimmunoblotting with antibodies against mouse macrophage iNOS and[¹²⁵I]-labeled protein A.

RNA isolation and Northern blot analysis Cells were taken out fromculture dishes directly by adding Ultraspec-II RNA reagent (BiotecxLaboratories Inc.) and total RNA was isolated according to themanufacturer's protocol. For Northern blot analyses, 20 μg of total RNAwas electrophoresed on 1.2% denaturing formaldehyde-agarose gels,electrotransferred to Hybond-Nylon Membrane (Amersham) and hybridized at68° C. with ³²P-labeled cDNA probe using Express Hyb hybridizationsolution (Clontech) as described by the manufacturer. The cDNA probe wasmade by PCR™ amplification using two primers (forward primer:5′-CTCCTTCAAAGAGGCAAAAATA-3′ (SEQ ID NO:1); reverse primer:5′-CACTTCCTCCAGGATGTTGT-3′ (SEQ ID NO:2) (Geller et al., 1993). Afterhybridization filters were washed two or three times in solution I(2×SSC, 0.05% SDS) for one hour at room temperature followed by solutionII (0.1×SSC, 0.1% SDS) at 50° C. for another hour. The membranes werethen dried and exposed with X-ray films (Kodak). The same filters werestripped and rehybridized with probes for GAPDH. The relative mRNAcontent for iNOS was measured after scanning the bands with a Biorad(Model GS-670) imaging densitometer.

Preparation of nuclear extracts and electrophoretic mobility shift assayNuclear extracts from stimulated or unstimulated astrocytes (1×107cells) were prepared as described (Dignam et al., 1983) with slightmodification. Cells were harvested, washed twice with ice-coldphosphate-buffered saline and lysed in 400 μl of buffer A (10 mM HEPES,pH 7.9, 10 mM KCl, 2 mM MgCl₂, 0.5 mM DTT, 1 mM PMSF, 5 μg/ml aprotinin,5 μg/ml pepstatin A, and 5 μg/ml leupeptin) containing 0.1% Nonidet P40for 15 min on ice, vortexed vigorously for 15 s, and centrifuged at14,000 rpm for 30 s. The pelleted nuclei were resuspended in 40 μl ofbuffer B (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mMMgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/mlpepstatin A, and 5 μg/ml leupeptin). After 30 min on ice, lysates werecentrifuged at 14,000 rpm for 10 min. Supernatants containing thenuclear proteins were diluted with 20 μl of modified buffer C (20 mMHEPES, pH 7.9, 20% (v/v) glycerol, 0.05 M KCl, 0.2 mM EDTA, 0.5 mM DTT,and 0.5 mM PMSF) and stored at −70° C. until use. Nuclear extracts wereused for the electrophoretic mobility shift assay using the NF-kB DNAbinding protein detection system kit (GIBCO/BRL), according to themanufacturer's protocol.

Construction of reporter plasmid, transfection and assay ofchloramphenicol acetyl transferase activity The CAT (chloramphenicolacetyl transferase) under the control of nitric oxide synthase promoter(iNOS) was created by subcloning 1.5 kb promoter from pGEM-NOS at Sph Iand Sal I restriction sites of pCAT-basic vector (Promega). Full lengthpromoter (Eberhardt et al., 1996) was amplified by using two primers(Forward: 5′-GAGAGTGTGCAAGTATTTGTAGGAG-3′ (SEQ ID NO:6) and reverse:5′-AAGGTGGCTGAGAAGTTTCA-3′ (SEQ ID NO:7)) from rat genomic DNA andcloned in pGEM-T vector (Promega) to produce pGEM-NOS. The clone wasconfirmed by restriction mapping and sequencing. The cells weretransfected by using the lipofectin (Life Technologies Inc., USA)method, as has been described in manufacturer's protocol, with 2 μg ofreporter plasmid. They were then stimulated 24 h after transfection andharvested after 14 h of stimulation. CAT activity was measured as hasbeen described.

Cell viability: Cytotoxic effects of all the inhibitors were determinedby the MTT assay measuring the metabolic activity of cells.

Results

Inhibitors of PP1/2A stimulate LPS-induced production of NO in ratprimary astrocytes: Rat primary astrocytes were cultured in serum-freeDMEM/F-12 in the presence of LPS and inhibitors of different proteinphosphatases. The concentration of NO as nitrite (a stable reactionproduct of NO with molecular oxygen) was measured in culturesupernatants after 24 h. It is evident from Table 13 that bacterial LPSat a concentration of 1.0 μg/ml induced the production of NO as nitriteby about 8 fold. L-NMA, a competitive inhibitor of NOS suppressedLPS-mediated nitrite secretion indicating that LPS-induced nitriterelease in rat primary astrocytes is dependent on NOS-mediated argininemetabolism (Table 13). Inhibitors of protein phosphatase (PP) 1/2A(calyculin A and microcystin), PP 2B (deltamethrin and fenvalerate) orprotein tyrosine phosphatase (dephostin and orthovanadate) alone wasneither stimulatory nor inhibitory to nitrite production in controlastrocytes. However, calyculin A and microcystin, when added with theaddition of LPS, potentially stimulated LPS-mediated induction ofnitrite production in astrocytes. In contrast, inhibitors of PP 2B(cypermethrin, deltamethrin and fenvalerate) had no effect onLPS-induced nitrite production in astrocytes indicating that stimulationof LPS-induced production of NO in astrocytes is specific for theinhibitors of PP 1/2A.

To understand the mechanism of stimulatory effect of inhibitors of PP1/2A on the LPS-mediated nitrite production in astrocytes, the effect ofthese inhibitors on the protein and mRNA level of inducible nitric oxidesynthase (iNOS) was examined. Rat primary astrocytes were incubated inserum-free DMEM/F-12 received calyculin A, microcystin or cantharidinalong with 1.0 μg/ml of LPS. After 24 h, concentration of nitrite wasmeasured in the supernatants as described in Example 6. Data were takenas the mean±S.D. of three different experiments. Cell homogenates wereelectrophoresed, transferred on nitrocellulose membrane andimmunoblotted with antibodies against mouse macrophage iNOS as describedin Example 6. After 6 h of incubation, cells were taken out directly byadding ultraspec-11 RNA reagent (Biotecx Laboratories Inc) to the platesfor isolation of total RNA, and northern blot analysis for iNOS mRNA wascarried out as described. Assays were conducted for control, LPS,LPS+calyculin A (1 nM), LPS+calyculin A (2 nM), LPS+microcystin (1 nM),LPS+microcystin (2 nM), LPS+cantharidin (200 nM), and LPS+cantharidin(400 nM). Consistent with the production of nitrite, western blotanalysis with antibodies against murine macrophage iNOS and northernblot analysis for iNOS mRNA of LPS-stimulated astrocytes clearly showedthat inhibitors of PP 1/2A (calyculin A, microcystin and cantharidin)enhanced the LPS-mediated induction of iNOS protein and mRNA.

Since the inhibitors of PP 1/2A stimulated the LPS-mediated induction ofiNOS, the inventor examined whether these inhibitors inhibited theactivities of PP 1/2A in LPS-treated astrocytes. The activities of PP1/2A were measured in homogenates after 30 min of incubation. Cellsincubated in serum-free DMEM/F-12 received different concentrations ofokadaic acid (0-20 nM) along with 1.0 μg/ml of LPS. After 30 min ofincubation, protein phosphatase activity was measured (nmol PI/mln/mg).Data was measured as the mean±S.D. of three different experiments. Cellsincubated in serum free DMEM/F-12 received different concentrations ofokadaic acid in the presence or absence of 1.0 μg/ml of LPS. After 24 hof incubation, nitrite concentrations (nmol/mg/24 h) were measured insupernatants. Data are mean±S.D. of three different experiments. Okadaicacid dose-dependently inhibited the activities of PP 1/2A and stimulatedthe LPS-mediated induction of iNOS protein and production of NO inastrocytes. In a similar manner, calyculin A also inhibited theactivities of PP 1/2A.

Cells incubated in serum-free DMEM/F-12 received differentconcentrations of okadaic acid (0, 1, 2, 4, 8, 15, and 20 nM) along with1.0 μg/ml of LPS. After 24 h of incubation, cell homogenates wereelectrophoresed, transferred on nitrocellulose membrane andimmunoblotted with antibodies against mouse macrophage iNOS as describedbefore.

TABLE 13 Effect of inhibitors of different protein phosphatases onLPS-induced production of NO in rat primary astrocytes Stimuli Nitrite(nmol/rag/24 h) Control  3.1 ± 0.3 LPS 28.2 ± 3.1 LPS + L-NMA (0.1 mM) 5.2 ± 0.4 LPS + Cypermethrin (1 nM) 27.6 ± 2.7 LPS + Deltamethrin (1nM) 26.8 ± 2.9 LPS + Fenvalerate (20 nM) 27.1 ± 2.1 LPS + Calyculin A (2nM) 67.8 ± 7.3 LPS + Microcystin (2 nM) 64.8 ± 7.2Astrocytes preincubated in serum-free DMEM/F-12 for 30 min with L-NMAand different inhibitors of protein phosphatases received LPS (1.0μg/ml). After 24 h of incubation, nitrite concentration in thesupernatants were measured as described under “Materials and Methods”.Data are expressed as the mean±S.D. of three different experiments.

Stimulation of LPS- and cytokine-induced production of NO by calyculin Ain C₆ glial cell Similar to primary astrocytes, proinflammatorycytokines and LPS induce the production of nitrite as well as theexpression of iNOS in rat C₆ glial cells (Feinstein et al., 1994a,Dobashi et al., 1997). Unlike astrocytes, neither LPS or cytokine(s)alone was not a sufficient inducer of NO production in rat C₆ glialcells (Feinstein et al., 1994a; Dobashi et al., 1997). A combination ofLPS and cytokines was required to induce the production of NO in C6glial cells (Feinstein et al., 1994a; Dobashi et al., 1997).

However, the addition of 2 nM calyculin A along with LPS and cytokinesto C₆ cells stimulated the expression of iNOS protein and the productionof NO (nmol/mg/24 hr) by more than three fold in C₆ glial cells. Samplestested included control, LPS+TNF-α, TNF-α+IFN-γ, TNF-α+IL-1β,LPS+TNF-α+calyculin A, TNF-α+IFN-γ+calyculin A, andTNF-α+IL-1β+calyculin A. After 24 h, concentration of nitrite wasmeasured in the supernatants as described. Data was measured as themean±S.D. of three different experiments. Cells incubated in serum-freeDMEM/F-12 received calyculin A along with LPS and cytokines. Cellhomogenates were electrophoresed, transferred on nitrocellulose membraneand immunoblotted with antibodies against mouse macrophage iNOS asdescribed. These results indicate that both in primary astrocytes and C6glial cells the inhibitors of PP 1/2A up regulate the cytokine-inducedexpression of iNOS and the production of NO.

Inhibition of LPS- and cytokine-induced NO production by inhibitors ofPP1/2A in rat peritoneal macrophages: Since inhibitors of PP 1/2Astimulated the LPS- and cytokine-induced NO production in rat primaryastrocytes and C6 glial cells, the effect of these inhibitors on NOproduction and expression of iNOS in rat resident macrophages wasexamined. Similar to astrocytes, inhibitors of PP 1/2A alone had noeffect on the induction of NO production. However, in contrast to thestimulation of NO production in astrocytes (Table 13), all threeinhibitors of PP1/2A (calyculin A, microcystin and cantharidin)inhibited the LPS-induced NO production in rat peritoneal macrophages.Cells in this study were incubated in serum-free DMEM/F-12 and receivedcalyculin A, microcystin or cantharidin along with 1.0 μg/ml of LPS.Samples tested included control, LPS, LPS+calyculin A (1 nM),LPS+calyculin A (2 n), LPS+microcystin (1 nM), LPS+microcystin (2 nM),LPS+cantharidin (200 nM), and LPS+cantharidin (400 nM). After 24 h,concentration of nitrite was measured (nmol/mg/24 h) in the supernatantsas described in Example 6. Data was measured as the mean±S.D. of threedifferent experiments. Cell homogenates were electrophoresed,transferred on nitrocellulose membrane and immunoblotted with antibodiesagainst mouse macrophage iNOS as described. After 6 h of incubation,cells were analyzed for iNOS mRNA by northern blotting technique asdescribed earlier. This decrease in NO production was accompanied by adecrease in iNOS protein and iNOS mRNA.

Okadaic acid, another very specific and potent inhibitor of PP 1/2A,also dose-dependently inhibited the LPS-mediated production of NO andexpression of iNOS protein in astrocytes. Cells incubated in serum-freeDMEM/F-12 received different concentrations of okadaic acid (0, 5, 10,15, and 20 nM in the presence or absence of 1.0 μg/ml of LPS. After 24 hof incubation, nitrite concentrations were measured in supernatants.Data was determined as the mean±SD. of three different studies. Cellsincubated in serum-free DMEM/F-12 received different concentrations ofokadaic acid along with 1.0 μg/ml of LPS. Samples tested includedcontrol, LPS, LPS+okadaic acid (1 nM), LPS+okadaic acid (2 nM),LPS+okadic acid (4 nM), LPS+okadic acid (8 nM), LPS+okadic acid (15 nM),LPS+okadic acid (20 nM). After 24 h of incubation, cell homogenates wereelectrophoresed, transferred on nitrocellulose membrane andimmunoblotted with antibodies against mouse macrophage iNOS as describedin Example 6.

Similar to macrophages, calyculin A was also found to inhibit the LPS-and cytokine-induced production of NO and the expression of iNOS proteinin the murine macrophage cell line RAW 264.7. Cells in this study wereincubated in serum-free DMEM/F-12 received calyculin A along with LPSand cytokines. Samples tested included control, LPS+TNF-α, TNF-α+IFN-γ,TNF-α+IL-1β, LPS+TNF-α+calyculin A, TNF-α+IFN-γ+calyculin A, andTNF-α+IL-1β+calyculin A. After 24 h, concentration of nitrite(nmol/mg/24 h) was measured in the supernatants as described in Example6. Data are mean±S.D. of three different studies. Cell homogenates wereelectrophoresed, transferred on antibodies against mouse macrophage iNOSas described. Taken together, these results indicate that PP 1/2Aactivities are required to induce iNOS gene expression in macrophages.

Differential effect of okadaic acid on iNOS promoter-derivedchloramphenicol acetyl transferase (CAT) activity in LPS-stimulated ratprimary astrocytes and macrophages Differential regulation of theinduction of iNOS mRNA and protein in astrocytes and macrophages by theinhibitors of PP 1/2A indicates that these inhibitors regulate thetranscription of iNOS gene differentially in these two different celllines. Therefore, to understand the effect of okadaic acid on thetranscription of iNOS gene, astrocytes and macrophages were transfectedwith a construct containing the iNOS-promoter fused to the CAT gene, andactivation of this promoter was measured after stimulating the cellswith LPS in the presence or absence of okadaic acid. Consistent with theeffect of okadaic acid on the production of NO and the expression ofiNOS in two different cell types, okadaic acid stimulated theLPS-induced CAT activity in astrocytes but inhibited the LPS-induced CATactivity in macrophages (FIG. 5) indicating that okadaic aciddifferentially regulates the transcription of iNOS gene in astrocytesand macrophages.

Effect of okadaic acid on the activation of NF-kB in rat primaryastrocytes and macrophages Inhibitors of PP 1/2A stimulated theinduction of iNOS in astrocytes but inhibited the induction of iNOS inmacrophages indicating that PP 1/2A transduce different signals in twodifferent cell types for the differential regulation of iNOS. Since theactivation of NF-kB is necessary for the induction of iNOS, tounderstand the basis of this differential regulation of induction ofiNOS by inhibitors of PP 1/2A, the effect of okadaic acid on theLPS-induced activation of NF-kB in astrocytes and macrophages wasexamined. Astrocytes and macrophages incubated in serum-free DMEM/F-12were treated with okadaic acid alone or together with LPS (1.0 μg/ml),and nuclear proteins were isolated. After 1 h of incubation, cells weretaken out to prepare nuclear extracts and nuclear proteins were used forthe electrophoretic mobility shift assay as described in Example 6.Samples assayed included nuclear extract of control cells, nuclearextract of LPS-treated cells, nuclear extract of LPS-treated cellsincubated with 100-fold excess of unlabelled oligonucleotide, nuclearextract of cells treated with okadaic acid (5 nM) alone, nuclear extractof cells treated with okadaic acid (10 nM) alone, nuclear extract of LPSand okadaic acid (5 nM) treated cells, and nuclear extract of LPS andokadaic acid (10 nM) treated cells. Activation of NF-kB was evaluated bythe formation of a distinct and specific complex in a gel-shiftDNA-binding assay. Treatment of astrocytes or macrophages with 1.0 μg/mlof LPS resulted in the activation of NF-kB.

This gel shift assay detected a specific band in response to LPS thatwas competed off by an unlabelled probe. Although okadaic acid alone atdifferent concentrations failed to induce the activation NF-kB inastrocytes yet okadaic acid alone induced the activation of NF-kB inmacrophages. However, in both astrocytes and macrophages, okadaic acidstimulated the LPS-induced activation of NF-kB.

Inhibitors of PP 1/2A stimulate the LPS-induced production of TNF-α inrat primary astrocytes and macrophages: Okadaic acid stimulated thetranscription of iNOS in astrocytes and attenuated the transcription ofiNOS in macrophages. However, in contrast, okadaic acid stimulated theactivation of NF-kB in both astrocytes and macrophages. Since theinduction of TNF-α also depends on the activation of NF-kB, the effectof okadaic acid on the LPS-induced production of TNF-α in astrocytes andmacrophages was studied. Consistent with the stimulatory effect ofokadaic acid on the LPS-induced activation of NF-kB, okadaic acidstimulated the LPS-induced production of TNF-α in both astrocytes andmacrophages (Table 14).

TABLE 14 Effect of inhibitors of PP 1 and PP 2A on LPS-inducedproduction of TNF-α in rat primary astrocytes and macrophages TNF-α(ng/24 h/mg protein) Stimuli Astrocytes Macrophages Control  0.3 ± 0.03 0.5 ± 0.06 LPS  5.8 ± 0.7  18.9 ± 52.3 LPS + Calyculin A (1 nM) 12.5 ±1.6 27.5 ± 3.1 LPS + CalyculinA(2 nM) 16.9 ± 2.1 31.2 ± 3.6 LPS +Okadaic acid (5 nM) 10.8 ± 1.2 24.3 ± 1.9 LPS + Okadaic acid (10 nM)14.6 ± 1.8 28.9 ± 3.4Cells preincubated in serum-free DMEM/F-12 with different concentrationsof okadaic acid for 30 min was stimulated with 1.0 jig/ml of LPS. After24 h of incubation, concentration of TNF-α was measured in supernatantsas described under “Materials and Methods”. Data are expressed as themean±S.D. of three different experiments.

Effect of inhibitors of PP1/2A on cell viability Astrocytes ormacrophages were incubated with different inhibitors of PP1/2A for 24 hand their viability was determined as measured by the MTT assay. None ofthe inhibitors at the concentrations used in this study decreased orincreased the viability of the cells. Therefore, stimulation of theexpression of iNOS in astrocytes and inhibition of the expression ofiNOS in macrophages by inhibitors of PP 1/2A are not due to any changein viability of either astrocytes or macrophages.

EXAMPLE 7 Cytokine-Mediated Induction of Ceramide Production isRedox-Sensitive

Materials and Methods

Reagents DMEM/F-12 and fetal bovine serum (FBS) were from GIBCO. HumanIL1β was from Genzyme. Mouse recombinant TNF-α was obtained fromBoehringer Mannheim, Germany. Diamide, buthione (S,R)-sulfoximine,N-acetyl cysteine, pyrrolidine dithiocarbamate were from Sigma.

Isolation and maintenance of rat primary microglia, oligodendrocytes andastrocytes Microglial cells were isolated from mixed glial culturesaccording to the procedure of Guilian and Baker (1986). Briefly, after 7days the mixed glial cultures were washed 3 times with DMEM/F-12containing 10% FBS and subjected to a shake at 240 rpm for 4 h at 37° C.on a rotary shaker. The floating cells were washed and seeded ontoplastic tissue culture flasks and incubated at 37° C. After 30 min thenon-attached cells (mostly oligodendrocytes) were removed by repeatedwashes and the attached cells were used as microglia. These cells wereseeded onto new plates for further studies. Ninety to ninety-fivepercent of this preparation was positive for nonspecific esterase, amarker for macrophages and microglia.

After 4 h shaking, the flasks were washed three times to remove thefloating cells. Medium with 10% FBS was added and flasks were subjectedto another cycle of shaking for 24 h at 250 rpm. The suspended cellswere spun at 200 g and incubated for 30 min in tissue culture dish. Thenon-attached or weakly attached cells (mostly oligodendrocytes) wereremoved and seeded onto polylysine coated dishes and cultured in mediumcontaining 1% FBS. Ninety-five to ninety-seven percent of these cellswere positive for galactocerebroside immunostaining.

Astrocytes were prepared from rat cerebral tissue as described byMcCarthy and DeVellis (1980). After 10 days of culture astrocytes wereseparated from microglia and oligodendrocytes by shaking for 24 h in anorbital shaker at 240 rpm. To ensure the complete removal of alloligodendrocytes and microglia, the shaking was repeated twice after agap of one or two days. Attached cells were trypsinized (1 mM EDTA and0.05% trypsin in 10 mM tris-buffer saline, pH 7.4) and distributed intoculture dishes. These cells when checked for the astrocyte marker glialfibrillar acidic protein (GFAP), were found to be 95 to 100% positive.C6 glial cells obtained from ATCC were also maintained in DMEM/F-12containing 10% FBS as indicated above.

Brain tissue Frozen and fixed X-adrenoleukodystrophy and multiplesclerosis brain tissues were obtained from Brain and Tissue Banks forDevelopmental Disorders, University of Maryland, Baltimore, Md. 21201.

Lipid extraction Approximately 1.0×10⁶ cells were exposed to differentcytokines in the presence or absence of antioxidants for differentperiods and lipids were extracted according to the methods described byWelsh (1996).

Quantification of sphingomyelin by HPTLC and densitometry Sphingomyelinwas separated from total lipid extracts by HPTLC (LPK-plates fromWhatman Labsales, USA) as described (Ganser et al., 1988) forphospholipids with the modification, that the plate was overrun for 30min during its development and was dried overnight in vacuum desicator.Sphingomyelin was quantitated by densitometric scanning using ImagingDensitometer (Model GS-670; Bio-Rad, USA) and software provided with theinstrument by the manufacturer.

Quantification of ceramide levels by diacylglycerol kinase assayCeramide content was quantified essentially according to Priess et al.using diacylglycerol (DAG) kinase and [γ-³²P]ATP (Priess et al., 1986).Briefly, dried lipids were solubilized in 20 μl of an octylβ-D-glucoside/cardiolipin solution (7.5% octyl β-D-glucoside, 5 mMcardiolipin in 1 mM DTPA) by sonication in a sonicator bath. Thereaction was then carried out in a final volume of 100 μl containing the20 μl sample solution, 50 mM imidazole HCl, pH 6.6, 50 mM NaCl, 12.5 mMMgCl₂, 1 mM EGTA, 2 mM dithiothreitol, 6.6 μg of DAG kinase, and 1 mM[γ-³²P]ATP (specific activity of 1-5×10⁵ cpm/nmol) for 30 min at roomtemperature. The labeled ceramide 1-phosphate was resolved with asolvent system consisting of methyl acetate:n-propanol:chloroform:methanol:0.25% KCl in water:acetic-acid(100:100:100:40:36:2). A standard sample of ceramide was phosphorylatedunder identical conditions and developed in parallel. Both standard andsamples had identical R_(F) value (0.46). Quantification of ceramide1-phosphate was carried out by autoradiography and densitometricscanning using Imaging Densitometer (Model GS-670; Bio-Rad, USA). Valuesare expressed either as arbitrary units (absorbance) or as percentchange.

Measurement of reduced glutathione (GSH) concentration in rat primaryastrocytes Concentration of intracellular GSH was measured using acolorimetric assay kit for GSH from RandD, USA. Briefly, 2×10⁶ cellswere homogenized in 500 μl of ice-cold 5% metaphosphoric acid andcentrifuged at 3000 g for 10 min. Supernatants were used to assay GSHusing 4-chloro-1-methyl-7-trifluoromethyl-quinolinium methylsulfate and30% NaOH at 400 nm.

Detection of DNA fragmentation Cells (1×10⁶) were pelleted in aneppendorf tube by centrifugation at 1,000 rpm for 5 min, washed with PBS(pH 7.4), resuspended gently in 50 μl of a lysis buffer [200 mM NaCl, 10mM Tris-HCl (pH 8.0), 40 mM EDTA (pH 8.0), 0.5% SDS, 400 ng RNase A/μl]and incubated at 37° C. for 1 h. The lysate received 200 μl of thedigestion buffer [200 mM NaCl, 10 mM Tris-HCl (pH 8.0), 0.5% SDS, 125 ngproteinase K/μl]. The contents were mixed by inversion several times andthen incubated at 50° C. for 2 h. An equal volume of a mixture phenol(pH 8.0), chloroform and isoamyl alcohol (25:24:1, v/v) was added,gently mixed for 10 min, and stored at room temperature for 2 min. Thetwo phases were separated by centrifugation at 3,000 rpm for 10 min. Theviscous aqueous phase was transferred to a fresh tube and thephenol/chloroform extraction was repeated. The aqueous phase wasextracted with an equal volume of chloroform and 1.0 M MgCl₂ was addedto the aqueous phase to a final concentration of 10 mM. The total DNAwas precipitated by the addition of 2 vols. of absolute ethanol withseveral inversions. DNA was pelleted by centrifugation at 3,000 rpm for15 min, washed with 70% ethanol and air-dried. The pellet was dissolvedin 25 μl of 10 mM Tris-HCl containing 1.0 mM EDTA (pH 8.0) andelectrophoresed in 1.8% agarose gel at 4° C. The gel was stained withethidium bromide and DNA-intercalated ethidium fluorescence wasphotographed on Polaroid film 665 (P/N) using an orange filter. To studyDNA fragmentation in banked human brain tissues, brain tissues weregently homogenized in 0.85 M sucrose buffer and nuclei were purifiedaccording to the procedure described previously (Lazo et al., 1991).Total genomic DNA was isolated from the nuclei and electrophoresed asdescribed.

Fragment end labeling of DNA on paraffin-embedded tissue sections of MSand X-AID brains Fragmented DNA was detected in situ by the terminaldeoxynucleotidyl transferase (TDT)-mediated binding of 3′-OH ends of DNAfragments generated in response to apoptotic signals, using acommercially available kit (TdT FragEL™) from Calbiochem, USA. Briefly,paraffin-embedded tissue slides were deparaffinized, rehydrated ingraded ethanol, treated with 20 μg/ml proteinase K for 15 min at roomtemperature, and washed prior to TdT staining. After TdT staining,sections were lightly counterstained with methyl green.

Results

N-Acetyl-L-cysteine (NAC) and pyrrolidine dititiocarbamate (PDTC) blockTNF-α- and IL-1β-induced degradation of sphingomyelin to ceramide inprimary rat astrocytes Rat primary astrocytes were cultured inserum-free media with TNF-α or IL-1β for different times to quantify theproduction of ceramide using diacylglycerol (DAG) kinase. Since DAGkinase phosphorylates both DAG and ceramide using [γ-³²P]ATP assubstrate, both lipids can be quantified in the same assay. Cells wereexposed to TNF-α (50 ng/ml) for different time intervals (0, 5, 15, 30,45, and 60 minutes). Lipids were extracted, and DAG and ceramidecontents were determined as described (i.e. optical density) in Example7. Results were measured as the mean±S.D. of three different studies. Itwas found that in astrocytes, the DAG content was much higher than theceramide content.

Stimulation of cells with TNF-α resulted in a time-dependent increase inthe production of ceramide (about 3 fold after 45 min). In contrast toinduction of ceramide production, the level of DAG, an activator ofprotein kinase C and acidic sphingomyelinase, was unchanged at differenttime points of stimulation. Similar to TNF-α (FIG. 6), stimulation ofastrocytes with IL-1β for different times also induced a significantincrease in the ceramide content (FIG. 7). Almost three to four foldincrease in ceramide production was found in astrocytes after 30 or 45min of exposure with TNF-α or IL-1β. This increase in ceramide wasparalleled by TNF-α- and IL-1β-induced decrease in sphingomyelin (FIG. 6and FIG. 7). Sphingomyelin concentration decreases of approximately 18to 25% could be observed as early as 15 min following treatment ofastrocytes (FIG. 6 and FIG. 7) and maximal effects of up to 45 to 50% SMhydrolysis were observed after 30 to 45 min of treatment with TNF-α orIL-1β. These results indicate that both TNF-α and IL-1β modulate thedegradation of sphingomyelin to produce ceramide, the putative secondmessenger of the sphingomyelin signal transduction pathway, in ratprimary astrocytes within a short time. Interestingly, it was found thattreatment of astrocytes with antioxidants like NAC or PDTC 1 h beforethe addition of TNF-α or IL-1β potentially blocked the decrease insphingomyelin as well as the increase in ceramide (FIG. 6 and FIG. 7)indicating that reactive oxygen species (ROS) are possibly involved incytokine-induced degradation of SM to ceramide.

TNF-α and IL-I˜decrease intracellular level of reduced glutathione (GSH)in rat primary astrocytes and NAC blocks this decrease Sinceintracellular level of GSH is an important regulator of the redox stateof a cell, to understand the relationship between induction of ceramideproduction and intracellular level of GSH in cytokine-stimulatedastrocytes, rat primary astrocytes were stimulated with TNF-α or IL-1βand the level of GSH was measured at different times. Cells preincubatedwith 10 mM NAC for 1 h received either TNF-α (50 ng/ml) or IL-1β (50ng/ml). At different time intervals (0, 15, 30, 45, 60, 75, and 90minutes), cells were scrapped off and GSH concentrations (100% value is210±18.5 nmol/mg protein) were measured as described Example 7.Measurements were done in duplicate. The stimulation of cells with TNF-αor IL-1β resulted in an immediate decrease in intracellular level of GSHwith the maximal decrease (66 to 70% of control) found within 15 to 30min of initiation of stimulation and with a further increase in time ofincubation, the level of GSH was found to be almost normalized (88 to95% of control at 90 min). These results show that cytokine stimulationinduces rapid, short-term production of oxidants which transientlydeplete GSH. However, intracellular level of GSH did not decrease whencells were stimulated with cytokines in presence of NAC indicating thatNAC inhibited the cytokine-induced degradation of SM to ceramide bymaintaining the normal levels of GSH.

Thiol-depleting agents induce the production of ceramide in rat primaryastrocytes Since NAC, a thiol antioxidant, blocked cytokine-mediateddepletion of intracellular level of GSH and breakdown of SM to ceramide,the effect of a thiol-depleting agents [diamide and buthione(S,R)-sulfoximine] on ceramide production was examined. Diamide reducesthe intracellular level of GSH by its oxidation to GSSG whereas buthione(S,R)-sulfoximine does so by blocking the synthesis of GSH (Shertzer etal., 1995; Akamatsu et al., 1997). Rat primary astrocytes werepreincubated with 10 mM NAC for 1 h received diamide (0.5 mM). Atdifferent time intervals (0, 15, 30, 45, and 60) cells were washed withHBSS and scrapped off. Lipids were extracted, and level of ceramide wasmeasured as described in the methods section. Ceramide levels wereexpressed as -fold change over the level at 0 minutes in this study.Results were measured as the mean±S.D. of three different studies. Atdifferent time intervals, intracellular level of GSH (100% value was210±18.5 mmol/mg protein) was measured as described in Example 7.Measurements were done in duplicate. Stimulating rat primary astrocyteswith diamide resulted in an immediate decrease in intracellular level ofGSH due to rapid consumption of intracellular GSH through itsnonenzymatic conversion to the oxidized dimer, GSSG (Shertzer et al.,1995) and marked induction of ceramide production (about 7 fold after 30min of stimulation) indicating that intracellular level of GSH is theimportant regulator of degradation of SM to ceramide. Consistent withthis conclusion, treatment of cells with NAC blocked diamide-mediateddecrease in GSH level and induction of ceramide production. Similar todiamide, buthione (S,R)-sulfoximine also decreased the level of GSH andinduced the production of ceramide. Thus the low GSH and/or highintracellular oxidant (ROS) levels induced by cytokines andthiol-depleting agents facilitated the induction of ceramide production,while the normal levels of GSH and/or low ROS induced or maintained bythe addition of NAC blocked the hydrolysis of sphingomyelin to ceramide.Taken together, these results demonstrate that the intracellular levelsof GSH and/or ROS regulate the extent to which sphingomyelin is degradedto ceramide and ceramide-mediated signaling cascades are transduced.

Aminotriazole and hydrogen peroxide induce the production of ceramide inrat primary astrocytes Inhibition of cytokine-mediated induction ofceramide production by antioxidants and induction of ceramide productionby thiol-depleting agents alone indicate the involvement of ROS in theinduction of ceramide production. Therefore, the effect of exogenousaddition of an oxidant, like H₂O₂, or endogenously produced H₂O₂ oninhibition of catalase with aminotriazole (ATZ), which inhibitsendogenous catalase to increase the level of H₂O₂, on the induction ofceramide production. Rat primary astrocytes were incubated with 5 mMaminotriazole or 0.5 mM H₂O₂ in presence or absence of 10 mM NAC. Atdifferent time intervals (0, 15, 30, 45, and 60 minutes), cells werewashed with HBSS and scrapped off. Lipids were extracted, and level ofceramide was measured as described in Example 7. Ceramide levels wereexpressed as -fold change over the level at 0 minutes in these studies.Results are measured as the mean±S.D. of three different studies. Thetime course of ceramide production in rat primary astrocytes followingthe addition of ATZ. Approximately 45 min following the addition of ATZ,ceramide generation increased more than 5-fold over baseline. However,pretreatment of cells with NAC blocked the ATZ-mediated increase inceramide production. Consistent with the increase in ceramide productionby ATZ, addition of exogenous H₂O₂ to astrocytes also induced theproduction of ceramide with the maximum increase of about 7-fold after15 min. These results clearly indicate that intracellular levels of ROSregulate the production of ceramide.

Inhibition of cytokine-mediated production of ceramide in rat primarymicroglia, oligodendrocytes and C6 glial cells by NAC Since NACinhibited the cytokine-mediated production of ceramide in rat primaryastrocytes, the inventor examined the effect of NAC on cytokine-mediatedinduction of ceramide production in rat primary oligodendrocytes,microglia and C₆ glial cells was examined. Rat primary microglia,oligodendrocytes and C₆ glial cells were preincubated with 10 mM NAC for1 h in serum-free DMEM/F-12 received TNF-α (50 ng/ml). Cells were washedwith HBSS and scrapped off at different intervals (0, 15, 30, 45, and 60minutes). Lipids were extracted, and ceramide content was measured asdescribed in Example 7. Ceramide levels were expressed as -fold changeover the level at 0 minutes in these studies. Results are mean±S.D. ofthree different studies. The addition of TNF-α to oligodendrocytes,microglia or C₆ glial cells induced the production of ceramide. Theincrease in ceramide in these cells ranges from 2.5 to 4-fold withhighest increase in glial cells and lowest in oligodendrocytes. Theceramide levels peaked in glial cells at 30 min following stimulationand 45 min of stimulation in oligodendrocytes and C₆ glial cells. Theseresults show that similar to astrocytes, the SM cycle is also present inmicroglia, oligodendrocytes and C₆ glial cells. Consistent with theeffect of NAC on the production of ceramides in astrocytes, thisantioxidant also potently blocked the TNF-α-induced production ofceramide in microglia, oligodendrocytes and C₆ glial cells indicatingthat ROS are also involved in cytokine-mediated ceramide production inthese cells was examined.

NAC inhibits TNF-α- and diamide-mediated apoptosis in rat primaryoligodendrocytes by increasing the intracellular level of GSH anddecreasing the production of ceramide: Since cytokine-mediated ceramideproduction is implicated in apoptosis of different cells including braincells (Brugg et al., 1996; Wiesner and Dawson, 1996), the effect of NACon TNF-α- as well as diamide-mediated apoptosis in rat primaryoligodendrocytes, as evidenced by electrophoretical detection ofhydrolyzed DNA fragments (“laddering”) was investigated. To understandthe role of intracellular level of GSH in inducing apoptosis, ratprimary oligodendrocytes were treated with TNF-α or with diamide, athiol-depleting agent. Cells preincubated with 10 mM NAC for 1 hreceived either diamide (0.5 mM) or TNF-α (50 ng/ml). Samples wereprepared for control cells, diamide, diamide+NAC, TNF-α, and TNF-α+NAC.After 12 h of incubation, cells were harvested and washed with PBS, andgenomic DNA was extracted and run on agarose gels as described in themethods section. Ten micrograms of DNA was loaded in each lane. Levelsof ceramide and GSH were measured in homogenates as described in Example7. Results were determined as the mean±S.D. of three different studies.Both TNF-α and diamide decreased the intracellular level of GSH,increased the level of ceramide and induced internucleosomal DNAfragmentation as evident from the typical ladder pattern that wasgenerated. Interestingly, blocking of diamide- as well as TNF-α-mediateddecrease in intracellular level of GSH by pre-treatment with NACinhibited the induction of ceramide formation and DNA fragmentationindicating that intracellular level of GSH regulate apoptosis inoligodendrocytes through ceramide formation.

DNA fragmentation in banked human brains with X-adrenoleukodystrophy(X-ALD) and multiple sclerosis (MS) In the CNS, apoptosis may play animportant pathogenetic role in neurodegenerative diseases such asischemic injury, and white matter diseases (Thompson, 1995; Bredesen,1995). Both X-adrenoleukodystrophy (X-ALD) and MS are demyelinatingdiseases with the involvement of proinflammatory cytokines in themanifestation of white matter inflammation. Several studiesdemonstrating the induction of proinflammatory cytokines at the proteinor mRNA level in MS patients' cerebrospinal fluid and brain tissue haveestablished an association of proinflammatory cytokines (TNF-α, IL-1β,IL-2, IL-6 and IFN-γ) with the inflammatory loci in MS (Maimone et al.,1991; Tepper et al., 1995; Rudick and Ransohoff, 1992). Recentdocumentation of the presence of TNF-α, IL-1β and IFN-γ in X-ALD brainhave revealed the neuroinflammatory character of this disease (Powers etal., 1992; McGuiness et al., 1997).

Therefore, to understand the underlying relationship among intracellularlevel GSH, level of ceramide and DNA fragmentation in cytokine-inflamedCNS of X-ALD and MS, the inventor measured the levels of GSH andceramide in homogenates was measured and the DNA fragmentation in nucleifrom brains of patients with X-ALD and MS was also studied. Genomic DNAisolated from nuclei of banked human brains was run on agarose gel andphotographed as described Example 7. Ten micrograms of DNA was loaded ineach lane. Levels of ceramide (optical density) and GSH (nmol/mgprotein) were measured in homogenates as described. Results weredetermined as the mean±S.D. of three different studies. In both X-ALDand MS brain homogenates, the level of GSH was lower (55 to 70% ofcontrol) and the level of ceramide was higher (2 to 3 fold) compared tothose found in control brains. Consistent with a lower level of GSH anda higher level of ceramide, genomic DNA isolated from nuclei of X-ALDand MS brains when run on agarose gels formed the typical ladder patternwhich was absent in both of the normal brains. To confirm apoptosis inbrain tissues of X-ALD and MS, paraffin-embedded tissue sections ofX-ALD and MS were stained with TdT-mediated fragment end labeling.Terminal deoxynucleotidyl transferase (TdT)-mediated end labeling of3′-OH ends of DNA fragments on paraffin-embedded tissue sections forcontrol, X-ALD and MS samples was carried out using a commerciallyavailable kit from Calbiochem, USA. Consistent with increased DNAfragmentation in isolated nuclei of X-ALD and MS, increased TdT stainingon brain sections of X-ALD and MS was observed as compared to those ofcontrols. These observations indicate that intracellular level of GSHare an important factor in cytokine-mediated degradation of SM toceramide and apoptosis in inflammatory demyelinating diseases like X-ALDand MS.

EXAMPLE 8 Lovastatin and Phenylacetate Inhibit the Induction of NitricOxide Synthase and Cytokines in Rat Primary Astrocytes, Microglia, andMacrophages

This study explores the role of mevalonate inhibitors in the activationof NF-kβ and the induction of inducible nitric oxide synthase (iNOS) andcytokines (TNF-α, IL-1β, and IL-6) in rat primary astrocytes, microglia,and macrophages.

Materials and Methods

Reagents Recombinant rat IFN-γ, DMEM/F-12 medium, FBS, and HBSS werefrom GIBCO-BRL (Gaithersburg, Md.). Human IL-1β was from Genzyme Corp.(Boston, Mass.). Mouse recombinant TNF-α was obtained from BoehringerMannheim (Mannheim, Germany). Lovastatin, mevastatin, and farnesylpyrophosphate were from BIO-MOL Res. Labs Inc. (Plymouth Meeting, Pa.).Mevalonate, cholesterol, ubiquinone, arginase and LPS (Escherichia coli,serotype 0111:B4) were from Sigma Chemical Co. (St. Louis, Mo.).N^(G)-methyl-L-arginine (L-NMA), FPT inhibitor II, and antibodiesagainst mouse macrophage iNOS were obtained from Calbiochem Corp. (LaJolla, Calif.). Immunoassay kits for TNF-α, IL-1β, and IL-6 wereobtained from R&D Systems, Inc. (Minneapolis; MN). NF-kβ DNA bindingprotein detection kit was from GIBCO-BRL. [γ-³²P]ATP (3,000 Ci/mmol) wasfrom Amersham Corp. (Arlington Heights, Ill.). [2-¹⁴C]acetate waspurchased from ICN Biomedicals Inc. (Irvine, Calif.). NaPA was preparedfrom phenylacetic acid (Sigma Chemical Corp.) and NaOH as described(Samid et al., 1992).

Induction of NO production in rat astrocytes, microglia, and C₆ glialcells Astrocytes were prepared from rat cerebral tissue as described byMcCarthy and DeVellis (McCarthy and DeVellis, 1980). Cells weremaintained in DMEM/F-12 medium containing 10% FBS. After 10 d ofculture, astrocytes were separated from microglia and oligodendrocytesby shaking for 24 h in an orbital shaker at 240 rpm. To ensure completeremoval of the oligodendrocytes and microglia, the shaking was repeatedtwice after a gap of 1 or 2 d. Cells were trypsinized, subcultured, andstimulated with LPS or different cytokines in serum-free DMEM/F-12medium. Microglial cells were isolated from mixed glial culturesaccording to the procedure of Guilian and Baker (Giulian and Baker,1986). In brief, on days 7-9 the mixed glial cultures were washed threetimes with DMEM/F-12 and subjected to a shake at 240 rpm for 2 h at 37°C. on a rotary shaker. The floating cells were washed, seeded ontoplastic tissue culture flasks, and incubated at 37° C. for 2 h. Theattached cells were removed by trypsinization and seeded onto new platesfor further studies. 90-95% of this preparation was found to be positivefor nonspecific esterase, a marker for macrophages and microglia. Forinduction of NO production, cells were stimulated with LPS or cytokinesin serum-free condition. C₆ glial cells obtained from American TypeCulture Collection (Rockville, Md.) were also maintained and inducedwith different stimuli as indicated above.

Isolation of rat macrophages and induction of NO production Residentmacrophages were obtained from rat by peritoneal lavage with sterileRPMI 1640 medium containing 1% FBS and 100 μg/ml gentamicin. Cells werewashed three times with RPMI 1640 at 4° C., and were maintained at 37°C. in a humidified incubator containing 5% CO₂ in air. Macrophages at aconcentration of 2×10⁶ ml in RPMI 1640 medium containing L-glutamine andgentamicin were added in volumes of 800 μl to a 35-mm plate. After 1 h,nonadherent cells were removed by washing, and 800 μl of serum-free RPMI1640 medium with various stimuli was added to the adherent cells. After24 h the culture supernatants were transferred to measure NO production.

Cell viability Cytotoxic effects of the inhibitors were deter-mined bymeasuring the cell viability by Trypan blue exclusion.

Assay for NO synthesis Synthesis of NO was determined by assay ofculture supernatants for nitrite, a stable reaction product of NO withmolecular oxygen. In brief, 400 μl of culture supernatant was allowed toreact with 200 μl of Griess reagent (Feinstein et al., 1994), and wasincubated at room temperature for 15 min. The optical density of theassay samples was measured spectrophotometrically at 570 nm. Freshculture media served as the blank in all experiments. Nitriteconcentrations were calculated from a standard curve derived from thereaction of NaNO₂ in the assay. Protein was measured (Bradford, 1976).

Incorporation of [¹⁴C]acetate into cholesterol Astrocytes grown in100-mm plates (˜80% confluency) and preincubated in serum-free mediawith lovastatin or NaPA for 8 h received [2-¹⁴C]acetate (10 μCi/plate).After 3 h, the cells were washed twice with PBS and scraped off. Thelipids were extracted with 1 ml 75% ethanol, and the ethanol extract wassaponified with 1 ml of 20% ethanolic KOH at room temperature. To this,2 ml of water was added, mixed, and extracted twice with 2 ml of hexane.The hexane extracts were dried under nitrogen, dissolved in 50 μl ofCHCl₃/MeOH (1:1), spotted on a TLC plate along with standard[³H]cholesterol, and run with hexane/ether/acetic acid (70:30:1). Theplate was then exposed to a photographic film that was stored at −20° C.and developed after 2 d. The lanes corresponding to standard cholesterolwere scraped and counted in 5 ml of scintillation fluid.

Immunoblot analysis for iNOS After a 24 h incubation in the presence orabsence of different stimuli, cells were scraped off, washed with Hank'sbuffer, and homogenized in 50 mM Tris-HCl (pH 7.4) containing proteaseinhibitors (1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5μg/ml leupeptin). After electrophoresis the proteins were transferredonto a nitrocellulose membrane, and the iNOS band was visualized byimmunoblotting with antibodies against mouse macrophage iNOS and[¹²⁵I]-labeled protein A.

RNA isolation and Northern blot analysis Cells were taken out fromculture dishes directly by adding Ultraspec-II RNA reagent (BiotecxLaboratories Inc., Houston, Tex.), and total RNA was isolated accordingto the manufacturer's protocol. For Northern blot analyses, 20 μg oftotal RNA was electrophoresed on 1.2% denaturing formal-dehyde-agarosegels, electrotransferred to Hybond-Nylon Membrane (Amersham Corp.), andhybridized at 68° C. with ³²P-labeled cDNA probe using Express Hybhybridization solution (Clontech, Palo Alto, Calif.) as described by themanufacturer. The cDNA fragment for iNOS was amplified by PCR™using twoprimers (forward primer: 5′-CTCCTTCAAAGAGGCAAAAATA-3′ (SEQ ID NO:1);reverse primer: 5′-CACTTCCTCCAGGATGTTGT-3′ (SEQ ID NO:2)), and wascloned in pGEM-T vector (Geller et al., 1993). The clone was confirmedby DNA sequencing, and the insert was used as probe. Afterhybridization, filters were washed two to three times in solution I(2×SSC, 0.05% SDS) for 1 h at room temperature, followed by solution II(0.1×SSC, 0.1% SDS) at 50° C. for another hour. The membranes were thendried and exposed with x-ray films (Eastman Kodak Co., Rochester, N.Y.).Same filters were stripped and rehybridized with probes for GAPDH. Therelative mRNA content for iNOS was measured after scanning the bandswith a BioRad Model GS-670; Richmond, Calif.) imaging densitometer.

Nuclear run-on assay For the measurement of gene transcription, nucleiwere prepared, and in vitro transcriptional activity was measured withnuclei (25×106 nuclei per assay) using 30 μCi of [α-³²P]-UTP (400Ci/mmol) as described (Caira et al., 1995). In brief, the filters wereprehybridized in 1 ml of hybridization buffer (50% formamide, 5×SSC, 1%SDS, 15% dextran sulfate, 1×Denhardt's solution, and 50 μg/ml heparin).After 24 h of prehybridization in the above buffer, hybridization wascarried out with the labeled RNAs (1.3×10⁵ cpm) at 42° C. for 60 h to 3μg of the immobilized plasmid pGEM-T as a control, or to plasmidscontaining inserts of rat glyceraldehyde-3-phosphate dehydrogenase, ratactin, and human iNOS cDNAs. The filters were washed twice in 2×SSC,0.1% SDS for 15 min at SAC, and twice in 0.5×SSC, 0.1% SDS for 15 min.Then the filters were treated with RNase buffer (300 mM NaCl, 10 mMTrisHCI, pH 7.4, 40 mM EDTA, 10 μg/ml RNase A, and 350 U/ml RNase T1) at37° C. for 30 min, in the same buffer without RNases for another 30 min,and were then autoradiographed.

Determination of TNF-α, IL-1β, and IL-6 in culture supernatants Cellswere stimulated with LPS in serum-free media for 24 h in the presence orabsence of lovastatin or NaPA, and concentrations of TNF-α, IL-1β, andIL-6 were measured in culture supernatants by using high-sensitivityenzyme-linked immunosorbent assay (R&D Systems, Inc.) according to themanufacturer's instructions.

Preparation of nuclear extracts and electrophoretic mobility shift assayNuclear extracts from stimulated or unstimulated astrocytes (1×10⁷cells) were prepared (Dignam et al., 1983) with slight modifications.Cells were harvested, washed twice with ice-cold PBS, and lysed in 400μl of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 2 mM MgCl₂, 0.5 mM DTT,1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/mlleupeptin) containing 0.1% Nonidet P-40 for 15 min on ice, vortexedvigorously for 15 s, and centrifuged at 14,000 rpm for 30 s. Thepelleted nuclei were resuspended in 40 μl of buffer B (20 mM Hepes, pH7.9, 25% [vol/vol] glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5mM DTT, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/mlleupeptin). After 30 min on ice, lysates were centrifuged at 14,000 rpmfor 10 min. Supernatants containing the nuclear proteins were dilutedwith 20 μl of modified buffer C (20 mM Hepes, pH 7.9, 20% [vol/vol]glycerol, 0.05 M KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF) andstored at −70° C. until use. Nuclear extracts were used for theelectrophoretic mobility shift assay using the NF-kβ DNA binding proteindetection system kit (GIBCO/BRL) according to the manufacturer'sprotocol.

Results

Inhibitors of mevalonate pathway inhibit LPS-induced expression of iNOSin primary astrocytes Both HMG-CoA reductase and mevalonatepyrophosphate decarboxylase are the rate-limiting enzymes of themevalonate pathway (Goldstein and Brown, 1990; Castillo et al., 1991).The inventor examined the effect of inhibitors of HMG-CoA reductase(lovastatin and mevastatin) and mevalonate pyrophosphate decarboxylase(NaPA) on the induction of iNOS and production of NO. Results in Table15 show that bacterial LPS at a concentration of 1.0 μg/ml induced theproduction of NO by about eightfold. Inhibition of NO production byarginase, an enzyme that degrades the substrate (Q-arginine) of NOS andL-NMA, a competitive inhibitor of NOS, indicates that LPS-induced NOproduction in astrocytes is dependent on NOS-mediated argininemetabolism (Table 15). Lovastatin or mevastatin alone was neitherstimulatory nor inhibitory to nitrite production in control astrocytes.Both the inhibitors, however, when added 8 h before the addition of LPS,potentially inhibited LPS-mediated induction of nitrite production inastrocytes. Only 25% inhibition in LPS-induced NO production was foundwhen lovastatin was added to the cells along with LPS, however, thedegree of inhibition increased with the increase in time ofpreincubation, with lovastatin reaching about 90% inhibition of NOproduction within 8-10 h of preincubation.

To understand the mechanism of inhibitory effect of these inhibitors onLPS-mediated nitrite production, we examined the effect on protein andmRNA levels of iNOS. Rat primary astrocytes were preincubated inserum-free media with different concentrations of lovastatin (5 or 10μM) or NaPA (2 or 5 mM) or a combination of 2 μM lovastatin and 2 mMNaPA for 8 h received 1.0 μg/ml of LPS. After 24 h, supernatants wereused for nitrite assay as described in the Methods section of thisexample. Data was measured as the mean±SD of three differentexperiments. Cell homogenates were electrophoresed, transferred ontonitrocellulose membranes, and immunoblotted with antibodies againstmouse macrophage iNOS as described in Methods. Samples tested includedcontrol, LPS, LPS+lovastatin (5 μM), LPS+lovastatin (10 μM), LPS+NaPA (2mM), LPS+NaPA (5 mM), and LPS+lovastatin (2 μM)+NaPA (2 mM). After 5 hof incubation, cells were taken out directly by adding ultraspec-II RNAreagent (Biotecx Laboratories Inc.) to the plates for isolation of totalRNA, and Northern blot analysis for iNOS mRNA was carried out asdescribed in Methods. Western blot analysis with antibodies againstmurine macrophage iNOS and Northern blot analysis for iNOS mRNA analysisof LPS-stimulated astrocytes clearly showed that both lovastatin andNaPA significantly inhibited the LPS-mediated induction of iNOS proteinand mRNA. A combination of lovastatin and NaPA at a dose lower than theone used individually almost completely inhibited LPS-induced productionof NO and expression of iNOS.

To gain further insight into the mechanism of the inhibitory effect oflovastatin and NaPA on LPS-mediated expression of iNOS mRNA, theinventor examined the influence of lovastatin and NaPA on the rate ofiNOS gene transcription, as measured by nuclear run-on assays. Ratprimary astrocytes preincubated in serum-free media with 10 μMlovastatin or 5 mM NaPA, or a combination of 2 μM lovastatin and 2 mMNaPA for 8 h received 1.0 μg/ml of LPS. After 4 h cells were taken out,and nuclei were collected for nuclear run-on assays. ³²P-labeled mRNAwas transcribed in vitro from isolated nuclei, and 1.3×10⁵ cpm of run-onproducts were hybridized to each blot as described in Methods. Theplasmids used were pGEM-T without any insert (negative control) orcontaining iNOS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), oractin cDNA inserts. Samples tested included control, LPS, LPS+lovastatin(10 μM), LPS+NaPA (5 mM), and LPS+lovastatin (2 μM)+NaPA (2 mM). LPSinduced the transcription of the iNOS gene in astrocytes, and thatpreincubation of cells with lovastatin or NaPA inhibited the relativerate of LPS-induced nuclear transcription of the iNOS gene. Consistentwith the inhibition of LPS-induced expression of mRNA, protein andactivity of iNOS by lovastatin and NaPA, the combination of lovastatinand NaPA completely inhibited the transcription of iNOS gene. Theseresults clearly indicate that lovastatin and NaPA inhibit LPS-inducedexpression of iNOS mRNA, protein and activity by inhibitingtranscription of the iNOS gene.

TABLE 15 Inhibition of LPS-induced NO Production in Rat PrimaryAstrocytes by Lovastatin and Mevastatin Nitrite Stimuli nmol/mg/24 h %Inhibition Control 2.9 ± 0.5 — LPS 25.3 ± 3.2  — LPS + arginase 5.9 ±0.8 87 LPS + L-NMA 5.5 ± 0.7 88 Lovastatin 2.9 ± 0.3 — Mevastatin 2.8 ±0.4 — LPS + lovastatin 5.2 ± 0.5 90 LPS + mevastatin 5.5 ± 0.5 88Astrocytes were cultured for 24 h in serum-free DMEM/F-12 with thelisted reagents; nitrite concentration in the supernatants was measuredas described in Methods. Arginase (100 U/ml) and L-NMA (0.1 mM) wereadded to the cells together with LPS (1.0 μg/ml), however, cellspreincubated with lovastatin (10 μM) or mevastatin (10 μM) for 8 hreceived LPS. Data are mean±SD of three different experiments.

To determine if the synergistic inhibitory effect of lovastatin and NaPAon LPS-induced iNOS expression in astrocytes could be explained solelyby inhibition of the mevalonate pathway, the inventor examined theincorporation of [2-¹⁴C]acetate into cholesterol. Cells preincubatedwith lovastatin or NaPA for 8 h received [2-¹⁴C]acetate for 3 h.Lovastatin (10 μM) and NaPA (5 mM) inhibited the synthesis ofcholesterol by 73±6.2 and 64±5.3%, respectively. The combination oflovastatin (2 μM) and NaPA (2 mM), however, caused 93±4.2% inhibition,indicating that lovastatin and NaPA affect cholesterol synthesis in anadditive fashion. Therefore, absence of complete inhibition of iNOS mRNAor protein by lovastatin or NaPA could be due to the absence of completeinhibition of the mevalonate pathway and depletion of mevalonatemetabolites.

Inhibition of LPS- and cytokine-induced production of NO by lovastatinin rat primary astrocytes Similar to LPS, different cytokines and theirseveral combinations are known to induce the expression of iNOS (Jaffreyand Synder, 1995; Mitrovic et al., 1994; Bo et al., 1994; Merrill etal., 1993). To examine whether cytokine-induced NO production is alsoinhibited by lovastatin, primary astrocytes were stimulated withdifferent combinations of LPS, TNF-α, IL-1β, and IFN-γ for 24 h, and theproduction of NO was measured. Rat primary astrocytes preincubated inserum-free media with 10 μM lovastatin for 8 h received differentcombinations of LPS and cytokines. After 24 h of incubation, productionof nitrite was measured in supernatants as described earlier. Data wasmeasured as the mean±SD of three different experiments. Cell homogenateswere analyzed for iNOS protein by immunoblotting technique as describedbefore. Concentration of different stimuli were as follows: LPS, 0.5μg/ml; TNF-α, 20 ng/ml; IL-1β, 50 ng/ml; IFN-γ, 50 U/ml. Samples thatwere assayed included control, LPS+TNF-α, LPS+IFN-γ, TNF-α+IL-1β,TNF-α+IFN-γ, LPS+TNF-α+lovastatin, LPS+IFN-γ+lovastatin,TNF-α+IL-1β+lovastatin, and TNF-α+IFN-γ+lovastatin. All the combinationsof LPS and cytokines significantly induced production of NO, however,addition of 10 μM lovastatin to astrocytes potently inhibited NOproduction and induction of iNOS protein, indicating that similar toLPS, cytokine-mediated expression of iNOS also involves the mevalonatepathway. Under similar conditions, lovastatin was also found to inhibitLPS- and cytokine-induced NO production in rat C₆ glial cells.

Inhibition of LPS-induced activation of NF-kβ and expression of iNOS bylovastatin and NaPA, and its reversal by farnesyl pyrophosphate in ratprimary astrocytes. Since activation of NF-kβ is necessary for inductionof iNOS (Xie et al., 1994; Kwon et al., 1995), to understand the basisof the inhibition of iNOS, the inventor examined the effect of theseinhibitors on LPS-induced activation of NF-kβ in astrocytes by gel-shiftDNA-binding assay. Rat primary astrocytes incubated in serum-free mediareceived 1.0 μg/ml of LPS. After 1 h of incubation, cells were taken outto prepare nuclear extracts, and nuclear proteins were used for theelectrophoretic mobility shift assay of NF-kβ as described in Methods.Samples tested included control, LPS, LPS-treated nuclear extract with25-fold excess of unlabeled probe, and LPS-treated nuclear extract witha 50-fold excess of unlabeled probe, respectively. Treatment ofastrocytes with 1.0 μg/ml of LPS resulted in activation of NF-kβ. Thisgel-shift assay detected a specific band in response to LPS that wascompeted off by an unlabelled probe. Lovastatin or NaPA alone atdifferent concentrations failed to induce NF-kβ.

In an additional experiment, cells preincubated in serum-free media with10 μM of lovastatin or 5 mM of NaPA for 8 h received 1.0 μg/ml of LPS.Samples tested included control, LPS, LPS+lovastatin (5 μM),LPS+lovastatin (10 μM), LPS+NaPA (2 mM), LPS+NaPA (5 mM). Bothlovastatin and NaPA, however, markedly inhibited LPS-induced activationof NF-kβ, indicating that inhibition of iNOS expression by lovastatinand NaPA is possibly due to inhibition of NF-kβ.

The inventor has demonstrated earlier that activation of NF-kβ isnecessary for iNOS expression in rat primary astrocytes, and that cAMPderivatives inhibit the expression of iNOS by inhibiting the activationof NF-kβ. To evaluate the possible mechanism of the effect of lovastatinand NaPA, or to determine whether reduced concentrations of end productsas opposed to intermediate products of the mevalonate pathway wereresponsible for the effects of lovastatin and NaPA, the inventorperformed rescue experiments with cholesterol, ubiquinone, mevalonate,and FPP. Cells preincubated in serum-free media with 10 μM of lovastatinor 5 mM of NaPA for 8 h received 1.0 μg/ml of LPS along with 100 μMmevalonate or 200 μM farnesyl pyrophosphate. After 24 h, supernatantswere used for nitrite assay as described in Methods. Data was measuredas the mean±SD of three different experiments. Samples tested includedcontrol, LPS, LPS+lovastatin, LPS+lovastatin+mevalonate,LPS+lovastatin+FPP, LPS+NaPA, LPS+NaPA+mevalonate, LPS+NaPA+FPP. After 5h of incubation, cells were analyzed for iNOS mRNA by Northern blottingtechnique as described earlier. After 1 h of incubation, cells weretaken out to prepare nuclear extracts, and nuclear proteins were usedfor the electrophoretic mobility shift assay of NF-kβ as described inMethods. The addition of 10 μm ubiquinone or cholesterol to astrocytesdid not prevent the inhibitory effect of lovastatin and NaPA. NF-kβ andiNOS were induced in the LPS, LPS+lovastatin+mevalonate,LPS+lovastatin+FPP, and LPS+NaPA+FPP treated cells. These observationssupport the possibility that depletion of intermediary products ratherthan end products of mevalonate pathway is responsible for the observedinhibitory effect of lovastatin or NaPA on LPS-induced iNOS expression.On the other hand, mevalonate or FPP substantially reversed theinhibitory effect of lovastatin on iNOS expression and NF-kβ activation.FPP, however, but not mevalonate, reversed the inhibitory effect ofNaPA, indicating that the use of mevalonate rather than its synthesis isthe prime target of the NaPA.

An inhibitor of Ras farnesyl protein transferase (FPT inhibitor II)inhibits LPS-induced expression of iNOS and activation of NF-kβ in ratprimary astrocytes. FPT inhibitor II selectively inhibits ras farnesylprotein transferase with the IC₅₀ of 75 nM. In whole cells, however,25-250 μM of FPT inhibitor II inhibits farnesylation of p21^(ras) by˜90% (Manne et al., 1995). Inhibition of LPS-induced expression of iNOSand activation of NF-kβ by NaPA and its reversal by FPP, but not bymevalonate, indicates a possible involvement of the farnesylationreaction in activation of NF-kβ and induction of iNOS. Sincefarnesylation is a necessary step for activation of p21^(ras), thecentral molecule upstream of the Raf/MAP kinase cascade, the inventorexamined the effect of FPT inhibitor II, an inhibitor of Ras farnesylprotein transferase, on LPS-mediated expression of iNOS and activationof NF-kβ in rat primary astrocytes. Rat primary astrocytes preincubatedin serum-free media with 100 μM or 200 μM FPT inhibitor II for 1 hreceived 1.0 μg/ml of LPS. After 24 h of incubation, supernatants wereused for nitrite assay as described in Methods. Data were determined asthe mean±SD of three different experiments. After 5 h of incubation,cells were analyzed for iNOS mRNA by Northern blotting technique asdescribed earlier. After 1 h of incubation, cells were taken out toprepare nuclear extracts, and nuclear proteins were used for theelectrophoretic mobility shift assay of NF-kβ as described in Methods.Samples tested included control, LPS, LPS+FPT inhibitor II (100 μM), andLPS+FPT inhibitor II (100 μM). Preincubation of cells for 1 h with 100or 200 μM FPT inhibitor II potentially inhibited LPS-induced activationof NF-kβ, expression of iNOS, and production of NO, demonstrating theimportance of p21^(ras) farnesylation in LPS-mediated activation ofNF-kβ and induction of iNOS in astrocytes.

Lovastatin and NaPA inhibit the LPS-induced expression of TNF-α, IL-β,and IL-6 in rat primary astrocytes. Activated astrocytes, the majorglial cell population of brain, are reported to secrete TNF-α, IL-1β,and IL-6 (Sharif et al., 1993). Since lovastatin and NaPA inhibitedLPS-induced expression of iNOS in astrocytes, the inventor examined theeffect of these two inhibitors on LPS-induced expression of TNFα, IL-1β,and IL-6. Rat primary astrocytes preincubated in serum-free media withdifferent concentrations of lovastatin (5 or 10 μM) or NaPA (2 or 5 mM),or a combination of 2 μM of lovastatin and 2 mM of NaPA for 8 h,received 1.0 μg/ml of LPS. Samples tested included control, LPS,LPS+lovastatin (5 μM), LPS+lovastatin (10 μM), NaPA (2 mM), LPS+NaPA (5mM), and LPS+lovastatin (2 μM)+NaPA (2 mM). After 5 h of incubation,cells were analyzed for TNF-α, IL-1β, and IL-6 mRNAs by Northernblotting technique as described earlier. Astrocytes preincubated withlovastatin or NaPA were stimulated with LPS. Concentrations of TNFα,IL-1β, and IL-6 were measured in the supernatants after 24 h ofincubation (Table 16), and the mRNA expression of these-cytokines wasexamined in the cells after 5 h of LPS stimulation. Bacterial LPSmarkedly induced the mRNA expression and production of respectivecytokines in astrocytes. Although lovastatin or NaPA alone had no effecton the production of cytokines, however, these two compounds stronglyinhibited LPS-induced production of TNF-α, IL-1β, and IL-6 in thesupernatants (Table 16). The decrease in cytokine production was alsoaccompanied by an inhibition of their mRNA expression, demonstratingthat lovastatin and NaPA down-regulate expression of all the inflamatorymediators (iNOS, TNF-α, IL-1β, and IL-6) in astrocytes.

TABLE 16 Inhibition of LPS-induced Production of NO, TNF-α, IL-1β, andIL-6 in Rate Primary Astrocytes, Microglia, and Macrophages byLovastatin and NaPA Production of Treatments NO or LPS + Cells cytokinesLPS only lovastatin LPS + NaPA Astrocytes NO 25.3 ± 3.2 5.2 ± 0.4 5.4 ±0.6 TNF-α  5.3 ± 0.8  0.3 ± 0.05  0.4 ± 0.06 IL-1β 10.4 ± 1.5 0.8 ± 0.11.1 ± 0.2 IL-6 136.5 ± 16.8 6.9 ± 0.9 7.6 ± 0.8 Microglia NO 81.2 ± 6.95.9 ± 0.4 6.9 ± 0.9 TNF-α 14.5 ± 2.1 0.9 ± 0.1 1.3 ± 0.2 IL-1β 28.2 ±3.4 2.1 ± 0.3 2.4 ± 0.2 IL-6 295.6 ± 33.5 7.8 ± 1.1 9.3 ± 1.2Macrophages NO 118.5 ± 12.5 7.2 ± 0.9 9.5 ± 0.7 TNF-α 18.6 ± 2.3 1.2 ±0.1 1.7 ± 0.2 IL-1β 34.6 ± 4.5 2.3 ± 0.3 3.1 ± .4  IL-6 350.0 ± 27.6 8.3± 0.6 10.2 ± 1.4 Cells preincubated with 10 μM lovastatin or 5 mM NaPA for 8 h inserum-free condition was stimulated with 1.0 μg/ml of LPS. After 24 h ofincubation, concentrations of NO, TNF-α, IL-1β, and IL-6 were measuredin supernatants as described in Methods. NO is expressed as nmol/24 h/mgprotein whereas TNF-α, IL-1β, and IL-6 are expressed as ng/24 h/mgprotein. Data are expressed as the mean±SD of three differentexperiments.

Inhibition of LPS-induced production of NO, TNF-α, IL-1β, and IL-6 inrat primary microglia and macrophages by lovastatin. Both macrophagesand microglia, important sources of NO and cytokines, activelyparticipate in the pathophysiologies of different inflamatory disorders.Since lovastatin and NaPA inhibited the LPS-induced production of NO,TNF-α, IL-1β, and IL-6 in astrocytes, the inventor also examined theeffect of these two compounds on LPS-stimulated production of NO, TNF-α,IL-1β, and IL-6 in rat primary microglia and macrophages (Table 16). Therate of production of NO and cytokines after LPS stimulation was muchhigher in both macrophages and microglia than in astrocytes. Similar toastrocytes, lovastatin or NaPA alone had no effect on the production ofNO and cytokines in macrophages and microglia. Both of these compounds,however, strongly inhibited the LPS-induced production of NO, TNF-α,IL-1β, and IL-6 in macrophages and microglia (Table 16). These studiesdemonstrate the importance of the mevalonate pathway in the LPS inducedproduction of NO, TNF-α, IL-1β, and IL-6 in astrocytes as well as inmicroglia and macrophages (Table 16). The inhibitors (lovastatin,mevastatin, or NaPA), cytokines (TNF-α, IL-1β, and IFN-γ), or LPS usedunder these experimental conditions had no effect on the viability ofastrocytes, microglia, or macrophages, measured by Trypan blueexclusion. Therefore, the conclusion drawn in this study is not due toany change in viability of the cells.

Discussion

Several lines of evidence presented herein clearly support theconclusion that inhibitors of HMG-CoA reductase (lovastatin ormevastatin) and NaPA reduce the induction of inflammatory mediators(iNOS, TNF-α, IL-1β, and IL-6) in rat astrocytes, microglia, andmacrophages, demonstrating the involvement of mevalonate metabolite(s)and farnesyl pyrophosphate in the induction of inflammatory mediators.This conclusion was based on the following observations: first,LPS-induced expression of iNOS, TNF-α, IL-1β, and IL-6, and activationof NF-kβ, was inhibited by lovastatin and NaPA; second, inhibitoryeffects of lovastatin and NaPA on LPS-mediated induction of iNOS andcytokines was not reversed by cholesterol and ubiquinone, end productsof mevalonate pathway, indicating that this inhibitory effect oflovastatin was not due to depletion of end products of mevalonatepathway; third, the reversal of inhibitory effect of lovastatin bymevalonate and FPP and that of NaPA by only FPP, but not by mevalonate,indicates that mevalonate and FPP are necessary compounds for LPS signaltransduction; fourth, inhibition of LPS-induced activation of NF-kβ andinduction of iNOS by FPT inhibitor II, an inhibitor of Ras farnesylprotein transferase, indicates that farnesylation of p21^(ras) or otherproteins is required for signal transduction in the LPS-inducedexpression of iNOS. Since iNOS, TNF-α, IL-1β, and IL-6 have beenimplicated in the pathogenesis of demyelinating and neurodegenerativediseases (Mitrovic et al., 1994; Merrill et al., 1993), the inventor'sresults provide a potentially important mechanism whereby inhibitors ofHMG-CoA reductase and mevalonate pyrophosphate decarboxylase mayameliorate neural injury. Inhibition of LPS-induced NF-kβ activation andiNOS expression by lovastatin, NaPA, and FPT inhibitor II indicates thatthe observed inhibition of iNOS expression is due to inhibition of NF-kβactivation.

Since mevalonate availability regulates the posttranslationalisoprenylation of many intracellular signaling proteins includingp21^(ras) (Goldstein and Brown, 1990), the observed inhibition of NF-kβactivation and induction of iNOS by lovastatin and NaPA may be due tothe decrease or lack of the isoprenylation of p21^(ras), that in turnleads to the lack of or abnormal signal transmission from receptortyrosine kinase to Raf/MAP kinase cascade, activation of NF-kβ, andinduction of iNOS. The prerequisite of Ras farnesylation in transductionof signals from receptor tyrosine kinase to Raf/MAP kinase cascadeindicates a possible role of metabolites of mevalonate pathway in themodulation of iNOS induction.

NO, a diffusible free radical, plays many roles as a signaling and as aneffector molecule in diverse biological systems including neuronalmessenger, vasodilation, and antimicrobial and antitumor activities(Nathan, 1992; Jaffrey and Synder, 1995). In the nervous system, NOappears to have both neurotoxic and neuroprotective effects, and mayhave a role in the pathogenesis of stroke and other neurodegenerativediseases, and in demyelinating conditions (e.g., multiple sclerosis,experimental allergic encephalopathy, X-adrenoleukodystrophy) associatedwith infiltrating macrophages and production of proinflamatory cytokines(Mitrovic et al., 1994; Merrill et al., 1993; Dawson et al., 1991). NOand peroxynitrite (reaction product of NO and O₂—) are potentially toxicmolecules to neurons and oligodendrocytes that may mediate toxicitythrough the formation of iron-NO complexes of iron-containing enzymesystems (Drapier and Hibbs, 1988), oxidation of protein sulfhydrylgroups (Radi et al., 1991), nitration of proteins, and nitrosylation ofnucleic acids and DNA strand breaks (Wink et al., 1991). Althoughmonocytes/macrophages are the primary source of iNOS in inflammation,LPS and other cytokines induce a similar response in astrocytes andmicroglia (Hu et al., 1995; Galea et al., 1992). NO derived frommacrophages, microglia, and astrocytes has been implicated in the damageof myelin-producing oligodendrocytes in demyelinating disorders likemultiple sclerosis and neuronal death during neuronal degeneratingconditions including brain trauma (Hu et al., 1995; Merrill et al.,1993). The studies described herein indicate that lovastatin and NaPA,alone or in combination, may represent a possible avenue of research fortherapeutics directed against cytokine- and nitric oxide-mediated braindisorders, particularly in demyelinating conditions.

EXAMPLE 9 Amelioration of Experimental Allergic Encephalomyelitis byInhibiting the Induction of NOS-2 and Proinflammatory Cytokines

Proinflammatory cytokines and inducible nitric oxide synthase (iNOS) areinvolved in the pathogenesis of experimental allergic encephalomyelitis(EAE), an animal model of multiple sclerosis (MS). In the present studythe inventor reports the use of N-acetylcysteine (NAC), NaPA andlovastatin as therapeutic agents for the amelioration of the autoimmunedemyelinatory disease in EAE. The development of demyelinating lesionsin EAE or MS is the result of a complex chain of events that involvesrecognition of specific antigen, T cell activation, recruitment ofnonspecific cells to the lesion, release of numerous cytokines andinflammatory mediators (e.g., NO) by resident glial cells andinfiltrating cells, which in turn leads to demyelination and CNS damage.NAC, a potent antioxidant, blocks the induction of iNOS and TNF-α in ratperitoneal macrophages, astrocytes and C₆ glioma. Lovastatin, aninhibitor of the rate limiting enzyme of the mevalonate pathway, hasalso been shown to block the induction of iNOS and proinflammatorycytokines (TNF-α, IL-1β and IL-6) in rat astrocytes, microglia andmacrophages. The inventor provides evidence that NAC, NaPA or lovastatininhibits the induction of proinflammatory mediators (TNF-α, IFN-γ andiNOS) in EAE central nervous system and also ameliorate the clinicalsymptoms of the EAE disease.

Materials and Methods

Reagents Female Lewis rats were purchased from Charles River BreedingLaboratories, Wilmington, Mass., USA. Myelin basic protein (MBP),complete Freund's adjuvant (CFA), N-acetylcysteine (NAC) and FITCconjugated anti-mouse IgG were purchased from Sigma Chemical Co., USA.Lovastatin was obtained from Calbiochem, USA.

Induction and clinical assessment of Experimental allergicencephalomyelitis (EAE) Female Lewis rats, 250-300 g, were housed in ratcages and provided with food and water ad libitum. Rats were inducedwith EAE by injecting intradermally 50 μg of myelin basic protein (MBP)per animal emulsified in complete Freund's adjuvant (CFA) into themedial footpad of each hind leg on day 1 followed by a booster injectionon the 7th day under ether anesthesia. Clinical symptoms in these ratsmanifest as an ascending paralysis resulting in death in most animals.The signs of EAE were scored as: (0) normal; (1) piloerection; (2) lossin tail tonicity; (3) hind leg paralysis; (4) paraplegia; and (5)moribund.

Drug Treatment Regiment Lovastatin, NAC or NaPA therapy was started onthe first day of immunization (day 1) and continued daily for theduration of the study. Lovastatin, NAC or NaPA was dissolved in salineand pH was adjusted to 7.0. One group of rats induced for EAE were giveni.p. injection of Lovastatin (2 mg/Kg body weight) and another group ofrats induced for EAE were given i.p. injection of NAC (150 mg/Kg bodyweight) or NaPA. One group of animals induced for EAE was left untreatedwhile another group of animals was not induced for EAE and used as thecontrol group.

Immunohistochemistry Brains were fixed in 10% buffered formalin(Stephens Scientific, Riverdale, N.J.). The tissues were embedded inparaffin and sectioned at 4 μm. Sections were then stained for variouscytokines and cell markers as described below. For single-labelimmunohistochemistry, sections were incubated with either anti-iNOSantibody (1:100, rabbit polyclonal, Calbiochem, LaJolla, Calif.) oranti-TNF-α antibody (1:100, rabbit polyclonal, Genzyme, Cambridge,Mass.) or anti-IFN-γ antibody (1:200, rabbit polyclonal, BiosourceInternational, Camarillo, Calif.) essentially as described for otherantibodies (Hooper et al., 1997). The tissue sections were furtherincubated with FITC conjugated anti-rabbit IgG (1:100, Sigma, St. Louis,Mo.), mounted with mounting media (EMS) and analyzed byimmunofluorescence microscopy (Olympus) using Adobe photoshop software.For immunofluorescent double-labeling, sections were incubated firstwith anti-iNOS (1:100) followed by macrophage marker ED1 (1:100, mousemonoclonal, Biosource International, Camarillo, Calif.). Anti-iNOS wasvisualized using TRITC conjugated anti-rabbit IgG—(1:100, Sigma, St.Louis, Mo.) and ED1 using FITC conjugated anti-mouse IgG (1:100, Sigma,St. Louis, Mo.). Negative control sections were incubated with FITC orTRITC conjugated IgG without the primary antibody.

Results

Expression of iNOS, TNF-α and IFN-γ in Lewis rat brain sections ofcontrol, EAE and drug-treated animals iNOS in the CNS of Lewis rats wasdetection by immunofluorescence. Brain sections of control, EAE, EAEtreated with NAC, EAE treated with NaPA or EAE treated with lovastatinwere immunostained for iNOS as described under materials and methods.Brain sections of rats with EAE show expression of iNOS protein as greenfluorescence in a significant number of cells as compared to control.Moreover, treatment of rats with NAC, lovastatin or NaPA, blocked theability of MBP to induce the expression of iNOS. NAC treatment seems tobe better than lovastatin or NaPA in blocking the induction of iNOS.

TNF-α in the CNS of Lewis rats was detected by immunofluorescence. Brainsections of control, EAE, EAE treated with NAC, EAE treated with NaPAand EAE treated with lovastatin were immunostained for TNF-α asdescribed under materials and methods. Similar to the expression ofiNOS, a good number of cells show the expression of TNF-α as greenfluorescence in EAE brain as compared to controls. Treatment with NAC,lovastatin or NaPA blocked the induction of TNF-α. In case of TNF-α,better inhibition was observed in brains of rats treated with lovastatinor NaPA.

Lewis rat brain sections were stained immunohistochemically for IFN-γ.Brain sections of control, EAE, EAE treated with NAC, EAE treated withNaPA and EAE treated with lovastatin were immunostained for IFN-γ asdescribed under materials and methods. NAC, lovastatin or NaPAtreatments also blocked the induction of IFN-γ in brains of animalschallenged with MBP. The demonstration of induction of TNF-α, IFN-γ andiNOS in brains of EAE shows a inflammatory disease process andinhibition of the induction of these cytokines in brains of rats treatedwith NAC, NaPA or lovastatin indicate that these drugs may be of valuein ameliorating the inflammatory disease process in EAE.

Co-localization of iNOS with macrophage/microglial marker ED1 Toidentify the cell type in the CNS of EAE which express iNOS, theinventor performed immunofluorescence double-labeling study using ED1, aspecific marker for macrophage/microglia cells of Lewis rat brainsections. Brain sections of control, EAE, EAE treated with NAC, EAEtreated with NaPA and EAE treated with lovastatin were immunostained foriNOS (red) and ED1 (green) as described under materials and methods.Co-expression of iNOS and ED1 that was visualized as yellow/orange wasseen only in EAE induced rat brain sections indicating thatmacrophage/microglia of EAE rat brain express iNOS. Animals induced forEAE and treated with NAC, NaPA or lovastatin showed expression of ED1,however, colocalization of ED1 with iNOS, as seen with EAE sections wasnot observed. In NAC-, NaPA or lovastatin-treated rat brain sections ED1expression was observed but not iNOS.

NAC and lovastatin protect against EAE disease in Lewis female ratsSince NAC, NaPA and lovastatin inhibited the expression of iNOS andproinflammatory cytokines in activated glial cells (astroglia andmicroglia) and macrophages and in the CNS of Lewis rats with EAE, theinventor examined the therapeutic potential of NAC, NaPA and/orlovastatin on the disease process of EAE. Administration of Lovastatinin Lewis female rats delays the onset of EAE disease symptoms. Data wastaken as average clinical disease scores where 0—normal; 1—piloerection;2—loss in tail tonicity; 3—hind leg paralysis; 4—paraplegia and5—moribund. Clinical symptoms of EAE appeared in MBP-treated Lewisfemale rats (n=9) from 7th day after first immunization. In this model,MBP induced a monophasic acute disease progression resulting in death on11th day, however, control animals receiving only complete Freund'sadjuvant did not show any disease symptoms. On the other hand treatmentof MBP-injected rats with NAC (n=9), lovastatin (n=9) or NaPA from firstday of immunization protected the rats from the severity of the disease.Both NAC and lovastatin-treated rats received milder clinical symptoms(highest clinical score was between 2 and 3) and specially lovastatinsignificantly delayed the onset of first of clinical symptom. Theseresults clearly demonstrate that NAC, NaPA and lovastatin provideprotection against neuroinflammatory disease of EAE.

Discussion

These studies clearly demonstrate that both NAC, NaPA and lovastatininhibit the expression of proinflammatory cytokines (TNF-α and IFN-γ)and iNOS in the CNS of Lewis rats with EAE and ameliorate theneuroinflammatory disease process in the central nervous system.Immunohistochemical results show a higher degree of expression of iNOS,tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in brains ofrats with acute monophasic EAE relative to the control animals. AlthoughNAC and lovastatin did not block the clinical symptoms of EAE completelyin Lewis rat they significantly reduced the severity of the disease. NACis a nontoxic drug which has been safely used in humans for more than 30years. Lovastatin is also approved as an cholesterol-lowering drug forhumans. Therefore, inhibition of the expression of proinflammatorycytokines and iNOS in the CNS of EAE rats and amelioration of the EAEdisease process by NAC and lovastatin indicates that these drugs mayhave therapeutic importance in the treatment of neuroinflammatorydiseases like MS.

EXAMPLE 10 Proinflammatory Cytokine-Mediated Apoptosis in DemyelinatingDiseases

In the present study, the inventor examined the possible involvement ofROS in cytokine-mediated activation of sphingomyelin breakdown andceramide formation in rat primary glial cells.

Materials and Methods

Reagent DMEM/F-12 and fetal bovine serum (FBS) were from LifeTechnologies, Inc. Human IL1-β was from Genzyme. Mouse recombinant TNF-αwas obtained from Boehringer Mannheim, Germany. Diamide, buthione(SR)-sulfoximine, N-acetylcysteine, and pyrrolidinedithiocarbamate werefrom Sigma.

Isolation and Maintenance of Rat Primary Microglia, Oligodendrocytes,and Astrocytes Microglial cells were isolated from mixed glial culturesaccording to the procedure of Guilian and Baker (1986). Briefly, after 7days the mixed glial cultures were washed 3 times with DMEM/F-12containing 10% FBS and subjected to a shake at 240 rpm for 4 h at 37° C.on a rotary shaker. The floating cells were washed and seeded ontoplastic tissue culture flasks and incubated at 37° C. After 30 min thenon-attached cells (mostly oligodendrocytes) were removed by repeatedwashes, and the attached cells were used as microglia. These cells wereseeded onto new plates for further studies. Ninety to ninety-fivepercent of this preparation was positive for nonspecific esterase, amarker for macrophages and microglia.

After 4 h shaking, the flasks were washed three times to remove thefloating cells. Medium with 10% FBS was added, and flasks were subjectedto another cycle of shaking for 24 h at 250 rpm. The suspended cellswere spun at 200×g and incubated for 30 min in tissue culture dish. Thenon-attached or weakly attached cells (mostly oligodendrocytes) wereremoved and seeded onto polylysine-coated dishes and cultured in mediumcontaining 1% FBS. Ninety-five to ninety-seven percent of these cellswere positive for galactocerebroside immunostaining.

Astrocytes were prepared from rat cerebral tissue as described byMcCarthy and DeVellis (1980). After 10 days of culture astrocytes wereseparated from microglia and oligodendrocytes by shaking for 24 h in anorbital shaker at 240 rpm. To ensure the complete removal of alloligodendrocytes and microglia, the shaking was repeated twice after agap of 1 or 2 days. Attached cells were trypsinized (1 mM EDTA and 0.05%trypsin in 10 mM Tris-buffered saline, pH 7.4) and distributed intoculture dishes. These cells when checked for the astrocyte marker glialfibrillar acidic protein were found to be 95-100% positive. C₆ glialcells obtained from ATCC were also maintained in DMEM/F-12 containing10% FBS as indicated above.

Brain Tissue Frozen and fixed X-adrenoleukodystrophy and multiplesclerosis brain tissues were obtained from Brain and Tissue Banks forDevelopmental Disorders, University of Maryland, Baltimore, Md. 21201.Two X-ALD brains were from 7- and 9-year-old males, and two MS brainswere from 30- and 33-year old females. Control brain for X-ALD studieswas from an 8-year-old male, and control brain for MS studies was from a30-year-old female.

Lipid Extraction Approximately 1.0×10⁶ cells were exposed to differentcytokines in the presence or absence of antioxidants for differentperiods, and lipids were extracted according to the methods described byWelsh (1996).

Quantification of Sphingomyelin by High Performance TLC and DensitometrySphingomyelin was separated from total lipid extracts by highperformance TLC (LHPK plates from Whatman) as described by Ganser et al.(1988) for phospholipids with the following modification: the plate wasoverrun for 30 min during its development and was dried overnight invacuum desiccator. Sphingomyelin was quantitated by densitometricscanning using Imaging Densitometer (model GS-670; Bio-Rad), andsoftware was provided with the instrument by the manufacturer.

Quantification of Ceramide Levels by Diacylglycerol Kinase AssayCeramide content was quantified essentially according to Priess et al.(1986) using diacylglycerol (DAG) kinase and [γ-³²P]ATP. Briefly, driedlipids were solubilized in 20 μl of an octyl β-D-glucoside/cardiolipinsolution (7.5% octyl β-D-glucoside, 5 mM cardiolipin in 1 mM DTPA) bysonication in a sonicator bath. The reaction was then carried out in afinal volume of 100 μl containing the 20-μl sample solution, 50 mMimidazole HCl, pH 6.6, 50 mM NaCl, 12.5 mM MgCl₂, 1 mM EGTA, 2 mMdithiothreitol, 6.6 μg of DAG kinase, and 1 mM [γ-³²P]ATP (specificactivity of 1-5×10⁵ cpm/nmol) for 30 min at room temperature. Thelabeled ceramide-1-phosphate was resolved with a solvent systemconsisting of methyl acetate:n-propyl alcohol:chloroform:methanol, 0.25%KCl in water:acetic acid (100:100:100:40:36:2). A standard sample ofceramide was phosphorylated under identical conditions and developed inparallel. Both standard and samples had identical R_(F) values (0.46).Quantification of ceramide-1-phosphate was carried out byautoradiography and densitometric scanning using Imaging Densitometer(model GS-670; Bio-Rad). Values are expressed either as arbitrary units(absorbance) or as percent change.

Measurement of GSH (Reduced Glutathione) and GSSG Oxidized GlutathioneConcentration of intracellular reduced GSH was measured using acalorimetric assay kit for GSH from R & D Systems. Briefly, 2×10⁶ cellswere homogenized in 500 μl of ice-cold 5% metaphosphoric acid andcentrifuged at 3000×g for 10 min. Supernatants were used to assay GSHusing 4-chloro-1-methyl-7-trifluoromethylquinolinium methylsulfate and30% NaOH at 400 nm. Concentration of GSSG was determined according tothe method of Griffith (1980) after derivatization with 2-vinylpyridinefor 30 min at room temperature.

Detection of DNA Fragmentation Cells (1×10⁶) were pelleted in anEppendorf tube by centrifugation at 1000 rpm for 5 min, washed withphosphate-buffered saline, pH 7.4, resuspended gently in 50 μl of alysis buffer (200 mM NaCl, 10 mM Tris-HCl, pH 8.0, 40 mM EDTA, pH 8.0,0.5% SDS, 400 ng of RNase A/μl), and incubated at 37° C. for 1 h. Thelysate received 200 μl of the digestion buffer (200 mM NaCl, 10 mMTris-HCl, pH 8.0, 0.5% SDS, 125 ng of proteinase K/μl). The contentswere mixed by inversion several times and then incubated at 50° C. for 2h. An equal volume of a mixture of phenol, pH 8.0, chloroform, andisoamyl alcohol (25:24:1, v/v) was added, gently mixed for 10 min, andstored at room temperature for 2 min. The two phases were separated bycentrifugation at 3000 rpm for 10 min. The viscous aqueous phase wastransferred to a fresh tube, and the phenol/chloroform extraction wasrepeated. The aqueous phase was extracted with an equal volume ofchloroform, and 1.0 M MgCl₂ was added to the aqueous phase to a finalconcentration of 10 mM. The total DNA was precipitated by the additionof 2 volumes of absolute ethanol with several inversions. DNA waspelleted by centrifugation at 3000 rpm for 15 min, washed with 70%ethanol, and air-dried. The pellet was dissolved in 25 μl of 10 mMTris-HCl containing 1.0 mM EDTA, pH 8.0, and electrophoresed in 1.8%agarose gel at 4° C. The gel was stained with ethidium bromide, andDNA-intercalated ethidium fluorescence was photographed on Polaroid film665 (P/N) using an orange filter. To study DNA fragmentation in bankedhuman brain tissues, brain tissues were gently homogenized in 0.85 Msucrose buffer, and nuclei were purified according to the proceduredescribed previously (Lazo et al., 1991). Total genomic DNA was isolatedfrom the nuclei and electrophoresed as described.

Fragment End Labeling of DNA on Paraffin-embedded Tissue Sections of MSand X-ALD Brains Fragmented DNA was detected in situ by the terminaldeoxynucleotidyltransferase-mediated binding of 3′-OH ends of DNAfragments generated in response to apoptotic signals, using acommercially available kit (TdT FragEL™) from Calbiochem. Briefly,paraffin-embedded tissue slides were deparaffinized, rehydrated ingraded ethanol, treated with 20 μg/ml proteinase K for 15 min at roomtemperature, and washed prior to terminal deoxynucleotidyltransferasestaining. After terminal deoxynucleotidyltransferase staining, sectionswere lightly counterstained with methyl green.

Results

NAC and PDTC Block TNF-α- and IL-1β-induced Degradation of Sphingomyelinto Ceramide in Primary Rat Astrocytes Rat primary astrocytes werecultured in serum-free media with TNF-α or IL-1β for different times toquantify the production of ceramide using diacylglycerol (DAG) kinase.Since DAG kinase phosphorylates both DAG and ceramide using [γ-³²P]ATPas substrate, both lipids can be quantified in the same assay. Ratprimary astrocytes were exposed to TNF-α (50 ng/ml) for different timeintervals (0, 5, 15, 30, 45 and 60 minutes). Lipids were extracted, andDAG and ceramide contents were determined as described under “Materialsand Methods.” Results were determined as the mean±S.D. of threedifferent studies. It was found that in astrocytes, the DAG content wasmuch higher than the ceramide content. Stimulation of cells with TNF-αresulted in a time-dependent increase in the production of ceramide(about 3-fold after 45 min). In contrast to induction of ceramideproduction, the level of DAG, an activator of protein kinase C andacidic sphingomyelinase, was unchanged at different time points ofstimulation.

In another experiment, rat primary astrocytes preincubated with either10 mM NAC or 100 μM PDTC for 1 h in serum-free DMEM/F-12 received TNF-α(50 ng/ml). At different time intervals (0, 15, 30, 45, and 60 minutes),cells were washed with HBSS and scraped off. Lipids were extracted, andlevels of ceramide (100% value is 4.51±0.1 nmol/mg protein) andsphingomyelin (100% value is 25.39±6.27 nmol/mg protein) were measuredas described under “Materials and Methods-” Results were measured as themean±S.D. of three different studies. TNF-α-induced degradation ofsphingomyelin to ceramide was inhibited by NAC and PDTC.

Similar to TNF-α, stimulation of astrocytes with IL-1β for differenttimes also induced a significant increase in the ceramide content. Ratprimary astrocytes preincubated with either 10 mM NAC or 100 μM PDTC for1 h in serum-free DMEM/F-12 received IL-1β (50 ng/ml). At different timeintervals (0, 15. 30, 45, and 60 minutes), cells were washed with HBSSand scraped off. Lipids were extracted, and levels of ceramide (100%value is 4.51±0.1 nmol/mg protein) and sphingomyelin (100% value is25.39±6.27 nmol/mg protein) were measured as described under “Materialsand Methods.” Results were measured as the mean±S.D. of three differentstudies. Almost 3-4-fold increase in ceramide production was found inastrocytes after 30 or 45 min of exposure with TNF-α or IL-1β. Thisincrease in ceramide was paralleled by TNF-α- and IL-1β-induced decreasein sphingomyelin. Sphingomyelin turnover of approximately 18-25% couldbe observed as early as 15 min following treatment of astrocytes, andmaximal effects of up to 45-50% SM hydrolysis were observed after 3045min of treatment with TNF-α or IL 1β.

These studies indicate that both TNF-α and IL-1β can modulate thedegradation of sphingomyelin to produce ceramide, the putative secondmessenger of the sphingomyelin signal transduction pathway, in ratprimary astrocytes within a short time. Interestingly, the inventorfound that treatment of astrocytes with antioxidants like NAC (10 mM) 1h before the addition of TNF-α or IL1β potentially blocked the decreasein sphingomyelin as well as the increase in ceramide, whereas 10 mMacetate had no effect on the degradation of SM to ceramide. Similar toNAC, another antioxidant PDTC also inhibited cytokine-mediateddegradation of SM to ceramide. These studies indicate that reactiveoxygen species (ROS) are possibly involved in cytokine-induceddegradation of SM to ceramide.

TNF-α and IL-1β Decrease Intracellular Level of Reduced Glutathione(GSH) in Rat Primary Astrocytes and NAC Blocks This Decrease Since theintracellular level of GSH is an important regulator of the redox stateof a cell, to understand the relationship between induction of ceramideproduction and intracellular level of GSH in cytokine-stimulatedastrocytes, cells were stimulated with TNF-α or IL-1β, and the level ofGSH was measured at different times (0, 15, 30, 45, 60, 75, and 90minutes). Rat primary astrocytes preincubated with 10 mM NAC for 1 hreceived either TNF-α (50 ng/ml) or IL-1β (50 ng/ml). At different timeintervals, cells were scraped off, and GSH concentrations (100% value is182.5±15.4 nmol/mg protein) were measured as described under “Materialsand Methods.” Results were measured as the mean±S.D. of three differentstudies. The stimulation of cells with TNF-α or IL-1β resulted in animmediate decrease in intracellular level of GSH with the maximaldecrease (66-70% of control) found within 15-30 min of initiation ofstimulation, and with a further increase in time of incubation, thelevel of GSH was found to be almost normal (88-95% of control at 90min). These studies indicate that cytokine stimulation apparentlyinduces rapid, short term production of oxidants which transientlydeplete GSH. However, the lack of decrease of GSH (FIG. 4) and lack ofhydrolysis of SM in the presence of NAC in the cytokine-treated cellsindicate that NAC inhibited the cytokine-induced degradation of SM toceramide by maintaining the normal levels of GSH.

Thiol-depleting Agents Induce the Production of Ceramide in Rat PrimaryAstrocytes Since NAC, a thiol antioxidant, blocked cytokine-mediateddepletion of intracellular levels of GSH and breakdown of SM toceramide, the inventor investigated the effect of thiol-depleting agents(diamide and buthione-(SR)-sulfoximine) on ceramide production. Diamidereduces the intracellular level of GSH by its oxidation to GSSG, whereasbuthione-(SR)-sulfoximine does so by blocking the synthesis of GSH(Shertzer et al., 1995; Akamatsu et al., 1997). Rat primary astrocytespreincubated with 10 mM NAC for 1 h received diamide (0.5 mM). Atdifferent time intervals (0, 15, 30, 45, and 60 minutes), cells werewashed with HBSS and scraped off. Lipids were extracted, and the levelof ceramide (100% value is 4.51±0.1 nmol/mg protein) was measured asdescribed under “Materials and Methods”. Results were measured as themean±S.D. of three different studies. Additionally, at different timeintervals (0, 15, 30, 45, and 60 minutes), intracellular level of GSH(100% value is 182.5±15.4 nmol/mg protein) was measured as describedunder “Materials and Methods.” Results were measured as the mean±S.D. ofthree different studies. Stimulating rat primary astrocytes with diamideresulted in an immediate decrease in intracellular level of GSH due torapid consumption of intracellular GSH through its nonenzymaticconversion to the oxidized dimer, GSSG (Shertzer et al., 1995), andmarked induction of ceramide production (about 7-fold after 30 min ofstimulation) indicating that intracellular level of GSH is the importantregulator of degradation of SM to ceramide. Consistent with this,treatment of cells with NAC blocked diamide-mediated decrease in GSHlevel and induction of ceramide production. Similar to diamide,buthione-(SR)-sulfoximine also decreased the level of GSH and inducedthe production of ceramide.

The inventor investigated the intracellular level of GSSG in astrocytestreated with TNF-α and diamide. Rat primary astrocytes were incubatedwith TNF-α (50 ng/ml) and diamide (0.5 mM), and at different timeintervals (0, 15, 30, 45, and 60 minutes) the intracellular level ofGSSG (100% value is 4.9±0.52 nmol/mg protein) was measured as describedunder “Materials and Methods.” Results were measured as the mean±S.D. ofthree different studies. In contrast to the decrease in intracellularlevel of GSH, both TNF-α and diamide increased the intracellular levelof GSSG. Thus it appears that the low GSH and/or high intracellularoxidant (ROS) levels induced by cytokines and thiol-depleting agentsfacilitated the induction of ceramide production, whereas the normallevels of GSH and/or low ROS induced or maintained by the addition ofNAC under these conditions blocked the hydrolysis of sphingomyelin toceramide. Taken together, these results demonstrate that theintracellular levels of GSH and/or ROS regulate the extent to whichsphingomyelin is degraded to ceramide, and ceramide-mediated signalingcascades are transduced.

Aminotriazole and Hydrogen Peroxide Induce the Production of Ceramide inRat Primary Astrocytes Inhibition of cytokine-mediated induction ofceramide production by antioxidants and induction of ceramide productionby thiol-depleting agents alone indicate the possible involvement of ROSin the induction of ceramide production. Therefore, the inventorexamined the effect of exogenous addition of an oxidant like H₂O₂ orendogenously produced H₂O₂ by inhibition of catalase with aminotriazole(ATZ), which inhibits endogenous catalase to increase the level of H₂O₂,on the induction of ceramide production. Rat primary astrocytes wereincubated with 5 mM aminotriazole (ATZ) or 0.5 mM H₂O₂ in presence orabsence of 10 mM NAC. At different time intervals (0, 15, 30, 45, and 60minutes), cells were washed with HBSS and scraped off. Lipids wereextracted, and the level of ceramide (100% value is 4.51±0.1 nmol/mgprotein) was measured as described under “Materials and Methods.”Results are mean±S.D. of three different studies. Approximately 45 minfollowing the addition of ATZ, ceramide generation increased more than5-fold over base line. However, pretreatment of cells with NAC blockedthe ATZ-mediated increase in ceramide production. Consistent with theincrease in ceramide production by ATZ, addition of exogenous H₂O₂ toastrocytes also induced the production of ceramide with the maximumincrease of about 7-fold after 15 min. These results clearly indicatethat intracellular levels of ROS regulate the production of ceramide.

Inhibition of Cytokine-mediated Production of Ceramide in Rat PrimaryMicroglia, Oligodendrocytes, and C₆ Glial Cells by NAC Since NACinhibited the cytokine-mediated production of ceramide in rat primaryastrocytes, the inventor examined the effect of NAC on cytokine-mediatedinduction of ceramide production in rat primary oligodendrocytes,microglia and C₆ glial cells. Rat primary microglia, oligodendrocytes,and C₆ glial cells preincubated with 10 mM NAC for 1 h in serum-freeDMEM/F-12 received TNF-α (50 ng/ml). Cells were washed with HBS andscrapped off at different intervals (0, 5, 30, 45, and 60 minutes).Lipids were extracted, and ceramide content (100% value for microglia,oligodendrocytes, and C₆ glial cells are 2.72±0.53, 3.37±0.32, 4.73±0.21nmol/mg protein, respectively) was measured as described under“Materials and Methods.” Results were determined as the mean±SD. ofthree different studies. The addition of TNF-α to microglia,oligodendrocytes, or C₆ glial cells induced the production of ceramide.The increase in ceramide in these cells ranges from 2.5- to 4-fold withhighest increase in glial cells and lowest in oligodendrocytes. Theceramide levels peaked in glial cells at 30 min following stimulationand 45 min of stimulation in oligodendrocyles and C₆ glial cells. Theseobservations show that similar to astrocytes, the SM cycle is alsopresent in microglia, oligodendrocytes and C₆ glial cells. Consistentwith the effect of NAC on the production of ceramides in astrocytes,this antioxidant also potently blocked the TNF-α-induced production ofceramide in microglia, oligodendrocytes, and C₆ glial cells indicatingthat ROS are also involved in cytokine-mediated ceramide production inthese cells.

NAC Inhibits TNF-α and Diamide-mediated Apoptosis in Rat PrimaryOligodendrocytes by Increasing the Intracellular Level of GSH andDecreasing the Production of Ceramide The inventor investigated theeffect of NAC on TNF-α as well as diamide-mediated apoptosis in ratprimary oligodendrocytes as evidenced by electrophoretical detection ofhydrolyzed DNA fray meets (“laddering”). To understand the role of theintracellular level of GSH in inducing apoptosis, the inventor treatedoligodendrocytes with TNF-α or with diamide, a thiol-depleting agent.Rat primary oligodendrocytes preincubated with 10 mM NAC for 1 hreceived either diamide (0.5 mM) or TNF-α (50 ng/ml). After 12 h ofincubation, cells were harvested and washed with phosphate-bufferedsaline, and genomic DNA was extracted and run on agarose gels asdescribed under “Materials and Methods.” Ten micrograms of DNA wasloaded in each lane. This study was repeated three times. Levels ofceramide (100% value is 3.37±0.32 nmol/mg protein) and GSH were measuredin homogenates as described under “Methods and Materials.” Results weremeasured as the mean±S.D. of three different studies. Both TNF-α anddiamide decreased the intracellular level of GSH, increased the level ofceramide, and induced internucleosomal DNA fragmentation as evident fromthe typical ladder pattern. Interestingly, blocking of the diamide- andTNF-α-mediated decrease in intracellular levels of GSH by pretreatmentwith NAC inhibited the induction of ceramide formation and DNAfragmentation indicating that intracellular levels of GSH may regulateapoptosis in oligodendrocytes through ceramide formation. To prove thisfurther, oligodendrocytes were treated with C₂-ceramide (acell-permeable ceramide analog) in the presence or absence of NAC. Ratprimary oligodendrocytes preincubated with 10 nM NAC for 1 h receivedC₂-ceramide. After 12 h of incubation, cells were harvested and washedwith phosphate-buffered saline, and genomic DNA was extracted and run onagarose gels as described under “Materials and Methods.” Ten microgramsof DNA was loaded in each lane. This study was repeated three times. Incontrast to the inhibitory effect of NAC on TNF-α-mediated apoptosis,NAC had no effect on C₂-ceramide-mediated apoptosis in oligodendrocytes.

DNA Fragmentation in Banked Human Brains with X-ALD and MS To understandthe underlying relationship among intracellular levels of GSH, levels ofceramide, and DNA fragmentation in cytokine-inflamed central nervoussystem of X-ALD and MS, the inventor measured the levels of GSH andceramide in homogenates and also studied the DNA fragmentation in nucleifrom brains of patients with X-ALD and MS. Regions surrounding plaquesof human brain white matter were used for DNA laddering and to measurethe levels of ceramide and GSH. Controls were age- and sex-matchedcontrols for X-ALD and MS, respectively. Since there was no plaque incontrol brains, the inventor used white matter of control brain for thisstudy. Genomic DNA isolated from nuclei of banked human brains was runon agarose gel and photographed as described under “Materials andMethods.” Ten micrograms of DNA was loaded in each lane. This study wasreproduced three times. The same amount of brain material (based onprotein concentration) was used to measure the level of ceramide asdescribed under “Materials and Methods-” Results were measured as themean±S.D. of three different studies. Concentrations of ceramide in theX-ALD and MS controls were 46.6±2.56 and 61.6±6.69 nmol/mg protein,respectively. The concentration of GSH was measured in homogenates asdescribed under “Materials and Methods.” Results for this experiment wasthe mean±S.D. of three different studies. In contrast to white mattersof control brains, white matters of both X-ALD and MS brains had severalplaque regions. In both X-ALD and MS brain homogenates, the level of GSHwas lower (55-70% of control), and the level of ceramide was higher (2-3fold) compared with those found in control brains. Consistent with alower level of GSH and a higher level of ceramide, genomic DNA isolatedfrom nuclei of X-ALD and MS brains when run on agarose gels formed thetypical ladder pattern, an indicator of apoptosis, which was absent inboth of the normal brains.

To confirm apoptosis in regions surrounding the plaques of white mattersof X-ALD and MS, paraffin-embedded tissue sections of X-ALD and MS werestained with terminal deoxynucleotidyltransferase-mediated fragment endlabeling. Terminal deoxynucleotidyltransferase-mediated end labeling of3′-OH ends of DNA fragments on paraffin-embedded tissue sections(control, X-ALD, and MS) was carried out using a commercially availablekit from Calbiochem. Regions surrounding plaques were used for thisstudy. Consistent with increased DNA fragmentation (apoptosis) inisolated nuclei of X-ALD and MS, the inventor observed increasedterminal deoxynucleotidyltransferase staining on brain sections of X-ALDand MS compared with those of controls. These biochemical andmorphological observations indicate that intracellular level of GSH maybe an important factor in cytokine-mediated degradation of SM toceramide and apoptosis in inflammatory demyelinating diseases like X-ALDand MS.

Discussion

The inventor shows that intracellular GSH plays a crucial role in thebreakdown of SM to ceramide, in that low GSH levels are required forceramide generation and high GSH levels inhibit production of ceramide.Inhibition of cytokine-mediated breakdown of SM to ceramide byantioxidants like N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate(PDTC) and induction of ceramide production by oxidants or pro-oxidantslike hydrogen peroxide, aminotriazole, diamide, and L-buthione-(SR)-sulfoximine clearly delineate a novel function of ROS andGSH in regulation of the first step of sphingomyelin signal transductionpathway. Moreover, decreased levels of GSH and increased levels ofceramide correlate with the DNA fragmentation in rat primaryoligodendrocytes as well as in the banked human brains from patientswith neuroinflammatory diseases (e.g., multiple sclerosis andX-adrenoleukodystrophy).

The present study underlines the importance of reactive oxygen speciesin cytokine-mediated degradation of sphingomyelin (SM) to ceramide.Treatment of rat primary astrocytes with tumor necrosis factor-α (TNF-α)or interleukin-1β led to marked alteration in cellular redox (decreasein intracellular GSH) and rapid degradation of SM to ceramide.Interestingly, pretreatment of astrocytes with N-acetylcysteine (NAC),an antioxidant and efficient thiol source for glutathione, preventedcytokine-induced decrease in GSH and degradation of sphingomyelin toceramide, whereas treatment of astrocytes with diamide, athiol-depleting agent, alone caused degradation of SM to ceramide.Moreover, potent activation of SM hydrolysis and ceramide generationwere observed by direct addition of an oxidant like hydrogen peroxide ora prooxidant like aminotriazole. Similar to NAC,pyrrolidinedithiocarbamate, another antioxidant, was also found to be apotent inhibitor of cytokine-induced degradation of SM to ceramideindicating that cytokine-induced hydrolysis of sphingomyelin isredox-sensitive. Besides astrocytes, NAC also blocked cytokine-mediatedceramide production in rat primary oligodendrocytes, microglia, and C₆glial cells. Inhibition of TNF-α- and diamide-mediated depletion of GSH,elevation of ceramide level, and DNA fragmentation (apoptosis) inprimary oligodendrocytes by NAC, and observed depletion of GSH,elevation of ceramide level, and apoptosis in banked human brains frompatients with neuroinflammatory diseases (e.g., X-adrenoleukodystrophyand multiple sclerosis) indicate that the intracellular level of GSH mayplay a critical role in the regulation of cytokine-induced generation ofceramide leading to apoptosis of brain cells in these diseases.

Changes in the cellular redox state toward either prooxidant orantioxidant conditions have profound effects on cellular functions.Several lines of evidence presented in this work indicate that the firststep of cytokine-induced sphingomyelin signal transduction pathway (i.e.breakdown of sphingomyelin to ceramide and phosphocholine) isredox-sensitive. First, cytokines like TNF-α and IL-1β decreasedintracellular GSH and induced the degradation of sphingomyelin toceramide in rat primary astrocytes, oligodendrocytes, microglia, and ratC₆ glial cells, and pretreatment of the cells with antioxidants like NACrestored the levels of GSH and blocked the degradation of sphingomyelinto ceramide. Second, depletion of endogenous glutathione by diamide orbuthione sulfoximine alone induces the degradation of sphingomyelin toceramide which is blocked by NAC. Third, the increase in intracellularH₂O₂ by the addition of exogenous H₂O₂ or by the inhibition ofendogenous catalase by aminotriazole induced the degradation ofsphingomyelin to ceramide which is also blocked by NAC. Fourth, besidesNAC, PDTC, an antioxidant but not the precursor of GSH (Laight et al.,1997), also inhibited the TNF-α and IL-1β-induced hydrolysis ofsphingomyelin to ceramide.

Over the years a number of sphingomyelinase activities have beenobserved in the cell. The major activities are the acid sphingomyelinasepresent in lysosomes, an enzyme with deficient activity in Niemann-Pickdisease (Spence, 1993), and plasma membrane-associatedmagnesium-dependent neutral pH optimal sphingomyelinase (Chatterjee,1993). In addition, a cytosolic magnesium-independent (Okazaki et al.,1994) and zinc-dependent acidic (Schissel et al., 1996) sphingomyelinasehave also been reported. The lysosomal acidic sphingomyelinase isbelieved to be responsible for degradation of sphingomyelin associatedwith turnover of membrane. The membrane-associated neutralsphingomyelinase is known to be activated in serum deprivation, TNF-α,and Fas-associated growth suppression and apoptosis (Tepper et al.,1995; Weigman et al., 1994). Although the studies reported here do notidentify the sphingomyelinase that is redox-sensitive, it is likely thatthe observed redox-sensitive hydrolysis of sphingomyelin incytokine-induced production of ceramide is mediated by the plasmamembrane-associated neutral sphingomyelinase.

The inventor's studies showing DNA fragmentation and increase inceramide and decrease in GSH in primary oligodendrocytes and bankedhuman brains with X-ALD and MS clearly indicate that intracellular redox(level of GSH) is an important regulator of apoptosis via controllingthe generation of ceramide. The inventor's conclusion is based on thefollowing observations. First, treatment of oligodendrocytes with TNF-αdecreased intracellular level of GSH, increased degradation of SM toceramide, and induced DNA fragmentation; however, pretreatment ofoligodendrocytes with NAC blocked the TNF-α-mediated decrease in GSHlevel, increase in ceramide level, and increase in DNA fragmentation. Incontrast, NAC had no effect on ceramide-mediated DNA fragmentation.Second, treatment of oligodendrocytes only with diamide, athiol-depleting agent, decreased intracellular level of GSH, increasedlevel of ceramide, and induced DNA fragmentations which are prevented bypretreatment of NAC, a thiol-replenishing agent. Third, the inventorobserved increased fragmentation of DNA in the white matter regionsurrounding plaques from patients with X-ALD and MS where the levels ofGSH and ceramide were lower and higher, respectively, compared withthose found in white matters of control human brains. These observationsindicate that maintenance of the thiol/oxidant balance is crucial forprotection against cytokine-mediated ceramide production and therebyagainst ceramide-induced cytotoxicity.

Observations described herein have demonstrated that ceramidepotentiates the cytokine-mediated induction of inducible nitric oxidesynthase in astrocytes and C₆ glial cells. Although ceramide by itselfdid not induce the expression of inducible nitric oxide synthase andproduction of NO, it markedly stimulated the cytokine-induced expressionof inducible nitric oxide synthase and production of NO indicating thatsphingomyelin-derived ceramide generation may be an important factor incytokine-mediated cytotoxicity in neurons and oligodendrocytes inneuroinflammatory diseases. The NAC, which has been used to block thecytokine-induced ceramide production in this study and to inhibitcytokine-mediated induction of inducible nitric oxide synthase, is anontoxic pharmaceutical drug that enters the cell readily and servesboth as a scavenger of ROS and a precursor of GSH, the majorintracellular thiol (Smilkstein et al., 1988). Therefore, the use ofreductants such as NAC or other thiol compounds may be beneficial inrestoring cellular redox and in inhibition of cytokine-mediatedinduction of inducible nitric oxide synthase and breakdown ofsphingomyelin thus reducing NO-mediated cytotoxicity as well asceramide-mediated apoptosis in neuroinflammatory diseases.

EXAMPLE 11 Lovastatin and Sodium Phenylacetate Normalize the Levels ofVery Long Chain Fatty Acids in Skin Fibroblasts ofX-Adrenoleukodystrophy

The inventor has observed that lovastatin and NaPA inhibit the inductionof nitric oxide synthase and proinflammatory cytokines (TNF-α, IL-1β andIL-6) in rat primary astrocytes, microglia and macrophages indicatingthat these drugs, alone or in combination, may represent a possibleavenue of research for therapeutics directed against cytokine- andNO-mediated brain disorders, particularly in demyelinating conditions.Lovastatin and NaPA have already been approved for medication/drugtrials for human diseases. In the current work the inventor providesevidence for the therapeutic intervention against pathognomonicaccumulation of VLCFA in X-ALD with these drugs.

Materials and Methods

Reagents DMEM, bovine calf serum and Hanks' buffered salt solution(HBSS) were from Gibco. [1-¹⁴C]Lignoceric acid was synthesized bytreatment of n-tricosanoyl bromide with K¹⁴CN as described previously(Hoshi and Kishimoto, 1973).

Enzyme assay for β-oxidation of lignoceric acid The enzyme activity of[1-¹⁴C]lignoceric acid β-oxidation to acetate was measured in intactcells suspended in HBSS. Briefly, the reaction mixture in 0.25 ml ofHBSS contained 50-60 μg of protein and 6 μM 1-¹⁴C]lignoceric acid. Fattyacids were solubilized with α-cyclodextrin and β-oxidation of[1-¹⁴C]lignoceric acid was carried out as described previously (Singh etal., 1984; Lazo et al., 1988).

Measurement of VLCFA in fibroblasts Fatty acid methyl ester (FAME) wasprepared as described previously by Lepage and Roy (1986) withmodifications. Fibroblast cells, suspended in HBSS, were disrupted bysonication to form a homogeneous solution. An aliquot (200 μl) of thissolution was transferred to a glass tube and 5 μg heptacosanoic (27:0)acid was added as internal standard and lipids were extracted by Folchpartition. Fatty acids were transesterified with acetyl chloride (200μl) in the presence of methanol and benzene (4:1) for 2 h at 100° C. Thesolution was cooled down to room temperature followed by addition of 5ml 6% potassium carbonate solution at ice-cooled temperature Isolationand purification of FAME were carried out as detailed by Dacremont etal. (1995). Purified FAME, suspended in chloroform, was analyzed by gaschromatograph GC-15A attached with chromatopac C-R3A integrator fromShimadzu Corporation.

Preparation of post-nuclear membrane and Western blot analysis TheMembranes were prepared as described previously (Contreras et al.,1996). Briefly, the post-nuclear fraction was diluted with an ice-coldsolution of 0.1 M sodium carbonate, 30 mM iodoacetamide, pH 11.5. After30 min of incubation at 4° C., the membranes were sedimented byultracentrifugation The sedimented membranes were electrophoresed in7.5% sodium dodecylsulfate-polyacrylamide gel, transferred to PVDFmembranes and immunoblotted with antibodies against ALDP as described(Contreras et al., 1996).

RNA isolation and Northern blot analysis Cultured skin fibroblasts weretaken out from culture flasks directly by adding Ultraspec-II RNAreagent (Biotecx) and total RNA was isolated according to themanufacturer's protocol. 20 μg of RNA from each sample waselectrophoretically resolved on 1.2% denaturing formaldehyde-agarosegel, transferred to nylon membrane, and crosslinked using UVStratalinker (Stratagene, La Jolla, Calif.). Full length ALDP cDNA waskindly provided by Dr. Patrick Aubourg, INSEAM, HospitalSaint-Vincent-de-Paul, Paris, France. ³²P-labeled cDNA probes wereprepared according to the instructions provided with Ready-To-Go DNAlabeling kit (Pharmacia Biotech). Northern blot analysis was performedessentially as described for Express Hyb Hybridization solution(Clontech, Palo Alto, Calif.) at 68° C. GAPDH cDNA probe was used asstandard for comparing hybridization signals.

Results

Inhibitors of mevalonate pathway stimulate the β-oxidation of lignocericacid in X-ALD fibroblasts. First, the inventor studied the effect ofmevalonate inhibitors (lovastatin, mevastatin and NaPA) on theβ-oxidation of lignoceric acid in control human skin fibroblasts. It isapparent from Table 17 that lovastatin, mevastatin and NaPA stimulatedthe β-oxidation of lignoceric acid in control human skin fibroblasts.Since the β-oxidation of lignoceric acid is impaired in X-ALD patients,the inventor studied the effect of these compounds on lignoceric acidβ-oxidation in cultured skin fibroblasts of X-ALD. Cultured skin X-ALDfibroblasts were incubated in serum-containing DMEM with differentconcentrations of lovastatin (0, 2, 4, 6, 8, and 10 μM) or NaPA (0, 1,2, 3, 4, and 5 mM) in the presence or absence of 2 μM lovastatin. Afterevery 24 h, medium was replaced with the addition of fresh reagents.Lignoceric acid β-oxidation was measured after 72 h in cell suspensionas described above. Values were measured as the mean±S.D. of threedifferent studies. Similar to control fibroblasts, these compounds alsostimulated lignoceric acid β-oxidation in X-ALD fibroblasts. Bothlovastatin and NaPA stimulated lignoceric acid β-oxidation in X-ALDfibroblasts in a dose-dependent manner. The highest dose of lovastatinfound to stimulate lignoceric acid β-oxidation (by 70%) was 5 μM whereasthe highest dose of NaPA found to stimulate lignoceric acid β-oxidation(by 40%) was 5 mM. However, a greater degree of stimulation (more thantwo-fold) was observed by a combination of lovastatin and NaPA even at adose lower than the one used individually. Higher doses of lovastatin(10-20 μM) or NaPA (1-20 mM) were cytotoxic to the X-ALD fibroblasts anddid not result in further significant stimulation. In the cell fattyacids are oxidized by mitochondrial and peroxisomal β-oxidation enzymesystems. The inventor examined the effect of etomoxir, an inhibitor ofmitochondrial β-oxidation, on the β-oxidation of fatty acids (Mannaertset al., 1979). Etomoxir had no effect on lovastatin- or NaPA-mediatedstimulation of lignoceric acid β-oxidation indicating that the observedstimulation of lignoceric acid β-oxidation was a peroxisomal function.

Modulation of cellular content of VLCFA in X-ALD fibroblasts bylovastatin and NaPA Since mevalonate inhibitors increased β-oxidation oflignoceric acid in control as well as X-ALD fibroblasts, the inventorexamined the effect of these compounds on the in situ levels of VLCFA inX-ALD fibroblasts. Cultured skin X-ALD fibroblasts were incubated inserum-containing DMEM with 5 μm lovastatin, 5 mM NaPA or the combinationof 2 μm lovastatin and 2 mM NaPA for different days (0, 3, 6, 9, 12, and15 days), and the ratios of C_(26:0)/C_(22:0) (A) and C_(24:0)/C_(22:0)(B) were measured as described. Values were determined as the mean oftwo different experiments. Treatment of X-ALD fibroblasts with 5 μm oflovastatin for different time periods (days) resulted in atime-dependent decrease in the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0). Within 12-15 days of treatment, the ratios ofC_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) in X-ALD fibroblasts decreasedto the normal level. Similar to lovastatin, NaPA also lowered the ratiosof C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) in X-ALD fibroblasts almostto the normal level after 15 days of treatment. However, consistent withthe higher degree of stimulation of lignoceric acid β-oxidation by acombination of lovastatin and NaPA, the same combination lowered theratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) to normal levelswithin 7 days. This decrease in the ratios of C_(26:0)/C_(22:0) andC_(24:0)/C_(22:0) was also associated with a decrease in the absoluteamounts of C_(24:0) and C_(26:0) whereas no significant change wasobserved in the levels of C_(22:0) (behenoic acid).

Normalization of the levels of VLCFA by lovastatin or NaPA in differentX-ALD cells with or without deletion of the X-ALD gene. Although theprecise function of ALDP, X-ALD gene product, in the metabolism of VLCFAis not known at the present time, however, accumulation of VLCFA inX-ALD cells with loss or mutations in ALDP and their normalizationfollowing transfection of cDNA of ALDP indicate a role of ALDP in themetabolism of VLCFA (Cartier et al., 1995). Therefore, the inventor nextattempted to examine whether lovastatin or NaPA were able to lower thelevels of VLCFA in X-ALD fibroblast cell lines with mutation or deletionof the X-ALD gene. Western blot analysis of post-nuclear membranefraction of X-ALD skin fibroblasts with antibodies against ALDP andNorthern blot analysis of X-ALD skin fibroblasts for ALDP mRNA werecarried out as described. ALDS2, ALDS3 and ALDS4 are X-ALD skinfibroblasts with mutation of the X-ALD gene, whereas ALDS5 and ALDS6 areX-ALD skin fibroblasts with deletion of the X-ALD gene. The status ofALDP mRNA or protein and the rate of β-oxidation of lignoceric acid(Table 18) in different X-ALD dibroblasts indicates that ALDS2, ALDS3and ALDS4 are X-ALD skin fibroblasts with mutation of the X-ALD gene,whereas ALDS5 and ALDS6 are X-ALD skin fibroblasts with deletion of theX-ALD gene. It is apparent from Table 18 that the treatment of X-ALDfibroblasts with lovastatin or NaPA or a combination of these stimulatedthe β-oxidation of lignoceric acid (55-80%) and normalized the ratios ofC_(26:0)/C_(22:0) indicating that these drugs are capable of loweringthe level of VLCFA in X-ALD fibroblasts to the normal levels,irrespective of mutation or deletion of the X-ALD gene, the candidategene for X-ALD.

TABLE 17 Lovastatin and NaPA stimulate the β-oxidation of lignocericacid in control human skin fibroblasts Lignoceric acid p-oxidationTreatment (pmol/h/mg protein) Control 570.2 ± 52.3 Lovastatin (5 μM) 945.7 ± 105.6 Mevastatin (5 μM) 889.6 ± 78.4 NaPA (5 mM) 826.2 ± 87.2Cells were treated for 72 h in serum-containing DMEM with the listedreagents; β-oxidation, of lignoceric acid was measured as described inSection 2. Medium was replaced after every 24 h with the addition offresh reagents. Data are mean±S.D. of three different studies.

TABLE 18 Effect of lovastatin and NaPA on (A) β-oxidation of lignocericacid and (B) the ratios of C_(26:0)/C_(22:0) and C_(24:0)/C_(22:0) incultured skin fibroblasts of X-ALD A. Lignoceric acid β-oxidation(pmol/h/mg protein) Cell line Control Lovastatin NaPA Lovastatin + NaPAALDS2 142.7 ± 15.7 223.5 ± 24.1 202.5 ± 17.4 274.6 ± 30.5 ALDS5 154.2 ±14.2 248.2 ± 26.2 211.5 ± 22.6 296.2 ± 25.6 ALDS6 132.4 ± 15.9 218.3 ±19.8 189.7 ± 21.2 250.1 ± 28.3 ALDS3 122.3 ± 11.7 201.3 ± 22.3 183.2 ±17.3 248.6 ± 29.6 ALDS4 118.5 ± 12.6 192.8 ± 20.5 178.9 ± 18.3 238.7 ±21.1 B. C_(26:0)/C_(22:0) C_(26:0)/C_(22:0) Control LovastatinLovastatin + NaPA Control Lovastatin Lovastatin + NaPA ALDS2 0.17 ±0.022 0.049 ± 0.01 0.04 ± 0.008 1.84 ± 0.25 1.25 ± 0.15 1.14 ± 0.15ALDS5 0.18 ± 0.025  0.055 ± 0.008 0.04 ± 0.007 1.94 ± 0.29 1.28 ± 0.211.18 ± 0.12 ALDS6 0.22 ± 0.034 0.058 ± 0.01 0.045 ± 0.008  2.01 ± 0.3 1.31 ± 0.18 1.21 ± 0.14 ALDS3 0.16 ± 0.024 0.045 ± 0.06 0.03 ± 0.0051.88 ± 0.21 1.26 ± 0.16 1.19 ± 0.25 ALDS4 0.19 ± 0.028 0.052 ± 0.070.036 ± 0.006  1.96 ± 0.23 1.29 ± 0.02 1.22 ± 0.15Cells were incubated in serum containing DMEM with 5 μM lovastatin, 5 mMNaPA or the combination of 2 μM lovastatin and 2 mM NaPA for 15 days,and the β-oxidation of lignoceric acid (A) and the ratios ofC_(26:0)/C_(22:0) and C_(24.0)/C_(22.0) (B) were measured as describedin Section 2. Results are mean±S.D. of three different studies. ALDS2,ALDS3 and ALDS4 are X-ALD skin fibroblasts with mutation of the X-ALDgene, whereas ALDS5 and ALDS6 are X-ALD skin fibroblasts with deletionof the X-ALD gene.Discussion

The present study underlines the importance of lovastatin, an inhibitorof 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, and thesodium salt of phenylacetic acid (NaPA), an inhibitor of mevalonatepyrophosphate decarboxylase, alone or in combination, in stimulating theβ-oxidation of lignoceric acid (C_(24:0)) and in normalizing thepathognomonic accumulation of saturated very long chain fatty acids(VLCFA) in cultured skin fibroblasts of X-adrenoleukodystrophy (X-ALD)in which the ALD gene is either mutated or deleted. The detailedmechanism leading to the decrease in the accumulation of VLCFA in X-ALDfibroblasts is not known, but is likely through the stimulation ofperoxisomal β-oxidation of VLCFA. In light of the fact that thesecompounds also inhibit the induction of proinflamrnatory cytokines andnitric oxide synthase in astrocytes and microglia, the ability oflovastatin and NaPA to normalize the pathognomonic accumulation of VLCFAin skin fibroblasts of X-ALD identify these drugs as possibletherapeutics for the neuroinflammatory disease process in X-ALD.

EXAMPLE 12 Lovastatin for X-Linked Adrenoleukodystrophy in Humans

The inventor has shown in animal studies that lovastatin, a3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and sodiumphenylacetate, an inhibitor of mevalonate pyrophosphate decarboxylase,inhibit the induction of inducible nitric oxide synthase andproinflammatory cytokines (tumor necrosis factor (alpha),interleukin-1(beta), and interleukin-6) involved in the pathogenesis ofneurologic damage in X-linked adrenoleukodystrophy. The inventor hasalso shown that lovastatin, sodium phenylacetate, and compounds thatincrease intracellular cyclic AMP and protein kinase A activitynormalize the levels of very-long-chain fatty acids in cultured skinfibroblasts from patients with childhood adrenoleukodystrophy andadrenomyeloneuropathy.

To demonstrate lovastatin's effectiveness in treating elevated VLCFAs inhuman patients, the inventor has treated seven patients from threefamilies with lovastatin for two to six months. The study was approvedby the institutional review board at the inventor's medical school, andthe patients provided informed consent. The diagnosis was established ineach case by clinical findings and documentation of elevated plasmalevels of very-long-chain fatty acids (C26:0) by two differentlaboratories. Each patient was treated with 20 mg of lovastatin per dayfor two weeks; the dose was increased to 40 mg per day if no adverseeffects were noted. Plasma very-long-chain fatty acids (C26:0) weremeasured periodically throughout the study. Adverse events andcompliance were assessed on the basis of the patients' reports and byperiodic measurement of plasma total cholesterol, creatine kinase,aspartate aminotransferase, and alanine aminotransferase.

One patient (Patient 4) was withdrawn from the study because ofpersistent diarrhea and a marked elevation of serum creatine kinaselevels. Another (Patient 5) discontinued treatment. The inventor'sresults (Table 19) show that plasma levels of very-long-chain fattyacids (C26:0) declined from their pretreatment values within one monthafter the initiation of lovastatin therapy in each patient and remainedlow and within the normal range for up to six months in the fivepatients who continued the treatment. Their lower post-treatmentcholesterol values (Table 19) provide evidence of compliance withtherapy. The short duration and small size of the study did not allowthe inventor to assess whether there was a clinical benefit.

TABLE 19 Effect of Lovastatin Therapy on Plasma Levels ofVery-Long-Chain Fatty Acids in Patients with X-LinkedAdrenoleukodystrophy Plasma Very-Long-Chain Fatty Acids† Plasma TotalCholesterol Age at Before AT 1 AT 2 AT 3 AT 4 AT 5 AT 6 Before AfterPatient Age Onset Clinical Treatment MO MO MO MO MO MO TreatmentTreatment‡ No. years Phenotype* micrograms per milliliter mg/dl 1 42 28Cerebral AMN 0.8 0.44 0.42 0.36 0.37 0.21 0.21 109 96 2 52 35 AMN and0.72 0.42 0.41 0.56 0.35 0.17 0.17 222 158 Addison's disease§ 3 55 45AMN 0.97 0.37 0.5 0.3  0.34 0.41 0.17 236 204 4 21 14 Adolescent 1.140.5 0.42 — 0.42 — — 165 167 cerebral ALD 5 25 — Presymptomatic 0.75 0.430.41 0.43 — — — 140 149 AMN¶ 6 44 — Heterozygous∥ 0.37 0.24 0.21 0.210.22 0.26 0.16 280 230 7 70 45 Heterozygous∥ 0.44 0.3 0.34 0.32 0.340.41 0.17 239 183 *AMN denotes adrenomyeloneuropathy, and ALDadrenoleukodystrophy. †The value in 50 normal control subjects was 0.24± 0.13 μg per milliliter. ‡Post-treatment total cholesterol levels wereobtained at six months for the patients who continued treatment(Patients 1, 2, 3, 6, and 7), at four months for Patient 4, and at threemonths for Patient 5. Patients 4 and 5 discontinued treatment.§Addison's disease is part of the spectrum of ALD. ¶Patient 5 hadnerve-conduction abnormalities consistent with AMN but no symptoms whenlast examined. ∥Patients 6 and 7 were women who carried one copy of themutant X gene. Patient 6 had no symptoms when last examined; patient 7had mild spasticity and paresthesia of both legs.

These results indicate that lovastatin treatment may represent a simple,safe, and effective way to reduce the accumulated plasma very-long-chainfatty acids in adult patients with X-linked adrenoleukodystrophy.

EXAMPLE 13 Treatment of Humans with Inhibitors of iNOS and Cytokines

The inhibitors, induction suppressors, induction enhancers, andstimulators or iNOS and/or proinflammatory cytokines of the presentinvention may be used in the treatment of cells and organisms such asmammals, including rodents and humans. These suppressors may be used toreduce the induction of iNOS and proinflammatory cytokines, reduce theaccumulation of VLCFAs, and treat neuroinflammatory diseases such asX-linked adrenoleukodystrophy and multiple sclerosis. As described inExample 12, lovastatin shows effectiveness in reducing the accumulationof very-long-chain fatty acids in human X-ALD patients. Any of thevarious inhibitors and/or induction suppressors described herein can beused in a human to treat any disease or disorder wherein a undesirableamount of iNOS and/or proinflammatory cytokines is acting to promotetissue damage. Dosages and combinations of inhibitors, suppressors,and/or other pharmaceuticals that may be used can be determined firstthrough an animal model of a particular disease or disorder, and thentested in a human population. Dosages may be optimized on an individualbasis, with routine experimentation by those of skill in the art.

* * *

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method of treating inflammation in cellsof a subject that does not have an elevated cholesterol level, to reducethe risk of cardiovascular disease or stroke, the method comprising: a)determining the level of inflammation in a subject, or in cells of thesubject, which subject does not have an elevated cholesterol level, bytesting for an indicator of inflammation; b) administering to cells ofthose subjects determined in step a) to have elevated level ofinflammation by virtue of such testing, an amount of an inhibitor of3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase that iseffective to reduce said inflammation and reduce the risk ofcardiovascular disease or stroke in said subject.
 2. The method of claim1, wherein said inhibitor HMG-CoA reductase is a statin.
 3. The methodof claim 2, wherein said statin is lovastatin or mevastatin.
 4. Themethod of claim 1, wherein the effective amount of said inhibitor isbetween about 0.25 mg/kg/day and 0.55 mg/kg/day.
 5. The method of claim1, wherein the inhibitor is comprised in a tablet.
 6. The method ofclaim 1, further defined as a method of treating inflammation in cellsof a subject determined to have elevated inflammation to reduce the riskof stroke in said subject.
 7. The method of claim 1, further defined asa method of treating inflammation in cells of a subject determined tohave elevated inflammation to reduce the risk of cardiovascular diseasein said subject.
 8. The method of claim 7, wherein the cardiovasculardisease is ischemia reperfusion.
 9. The method of claim 1, wherein thecells or the subject has been determined to have elevated inflammationby virtue of the presence of an indicator of NF-κβ activation.
 10. Themethod of claim 9, wherein the indicator of NF-κβ activation is elevatediNOS.
 11. The method of claim 9, wherein the indicator of NF-κβactivation is elevated proinflammatory cytokines.
 12. The method ofclaim 6, wherein the cells or the subject has been determined to haveelevated inflammation by virtue of the presence of an indicator of NF-κβactivation.
 13. The method of claim 12, wherein the indicator of NF-κβactivation is elevated iNOS.
 14. The method of claim 12, wherein theindicator of NF-κβ activation is elevated proinflammatory cytokines. 15.The method of any one of claims 1 through 14, wherein said subject is ahuman and the cells are human cells.
 16. The method of claim 1, whereinthe subject is a rodent and the cells are rodent cells.
 17. The methodof any one of claims 1 through 14, wherein the subject does not have anelevated lipid level.